Method of inhibiting tau phosphorylation

ABSTRACT

A method of inhibiting phosphorylation of the tau protein and/or a TLR4-mediated immune response is disclosed. The method contemplates administering to cells in recognized need thereof such as cells of the central nervous system an effective amount of a of a compound or a pharmaceutically acceptable salt thereof that binds to a pentapeptide of filamin A (FLNA) of SEQ ID NO: 1, and contains at least four of the six pharmacophores of FIGS.  35 - 40.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from application Ser. No. 61/789,180that was filed on Mar. 15, 2013, and application Ser. No. 61/671,235that was filed on Jul. 13, 2012, whose disclosures are incorporatedherein by reference.

TECHNICAL FIELD

The present invention contemplates a method of central nervous system(CNS) treatment to inhibit the formation of hyperphosphorylated tauprotein and the use of a contemplated compound in the manufacture of amedicament for inhibiting tau protein hyperphosphorylation that can leadto pathological formation of neurofibrillary tangles (NFTs). The methodand use also lead to the enhancement of function of one or more of thealpha-7 nicotinic acetylcholine receptor (α7nAchR), the insulin receptorand the N-methyl-D-aspartate receptor.

BACKGROUND ART

The microtubule-associated protein tau (MAPT) occurs mostly in axons andin lesser amounts in astrocytes and oligodendrocytes, and stabilizesneuronal microtubules for their role in the development of cellprocesses, establishing cell polarity and intracellular transport. Asingle gene encodes a tau protein with an open reading frame that canencode 758 amino acid residues. Tau is listed in theUniProtKB/Swiss-Prot data base under the designation P10636. At leastnine alternative splicing isoforms are recognized in theUniProtKB/Swiss-Prot data base.

Early work by Goedert and co-workers identified six isoforms thatcontain 352 to 441 amino acid residues [Mandelkow et al., Trends in CellBiology, 8:425-427 (1998); see also, Johnson et al., J. Cell Sci, 117(24):5721-5729 (2004)]. The numbering of the amino acid residue sequenceand the phosphorylation positions referred to herein is done in linewith the human tau isoform referred to as “htau 40” in Goedert et al.,Neuron 3:519-526 (1989). That 441 residue tau isoform is also referredto as Tau-4 or Tau-F in the UniProtKB/Swiss-Prot data base in which itis given the designation P10636-8. The amino acid residue sequence ofTAU-4 (P10636-8, htau 40) is shown in SEQ ID NO: 2.

Tau is a substrate for a number of kinase enzymes [Johnson et al., JCell Sci, 117(24):5721-5729 (2004)]. Phosphorylation at serine andthreonine residues in S-P or T-P motifs by proline-directed proteinkinases (PDPK1: CDK1, CDK5, GSK3, MAPK) and at serine residues inK-X-G-S motifs by MAP/microtubule affinity-regulating kinase (MARK1 orMARK2) are frequently found.

An enzyme of that group, glycogen synthase kinase 3β (GSK3β), can be apredominant tau kinase [Cho et al., J. Neurochem, 88:349-358 (2004)].GSK3β can phosphorylate unprimed sites that are in proline-rich regions(Thr-181, Ser-184, Ser-262, Ser-356 and Ser-400) or unprimed sites(Ser-195, Ser-198, Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-262,Ser-356 and Ser-404) where a serine or threonine is prephosphorylated byanother protein kinase (e.g., A-kinase) at a site that is located fouramino acid residues C-terminal to the GSK313 site [Cho et al., J.Neurochem, 88:349-358 (2004); Wang et al., FEBS Lett, 436:28-34 (1998)].

The normophosphorylated form of the protein is a microtublule-associatedprotein that stimulates and stabilizes microtubule assembly. Thatnormophosphorylated form typically contains two-three moles of phosphateper mole of protein [Kickstein et al., Proc Natl Acad Sci, USA,107(50):21830-21835 (2010)].

Multiply phosphorylated (hyper-phosphorylated) tau proteins; i.e., tauproteins that contain more than the normophosphorylated number ofphosphate groups, can result in the formation of neurofibrillary tanglesthat are associated with several pathological conditions that arereferred to collectively as tauopathies. For example, tauphosphorylation levels in Alzheimer's disease patients are three- tofour-fold higher than the number of phosphate groups present in thenormophosphorylated molecule [Kickstein et al., Proc Natl Acad Sci, USA,107(50):21830-21835 (2010)].

Increasing evidence suggests that neuroinflammation is a common featureof tauopathies. Thus, activated microglia are found in the postmortembrain tissues of various human tauopathies including Alzheimer's disease(AD), frontotemporal dementia (FTD), progressive supranuclear palsy andcorticobasal degeneration [Gebicke-Haerter, Microsc Res Tech, 54:47-58(2001); Gerhard et al., Mov Disord, 21:89-93 (12006); Ishizawa et al.,JNeuropathol Exp Neurol, 60:647-657 (2001)].

Induction of systemic inflammation via administration of the Toll-likereceptor 4 (TLR4) ligand, lipopolysaccharide (LPS), significantlyinduces MAPT (tau) hyperphosphorylation in a triple transgenic mousemodel of AD [Kitazawa et al., J Neurosci, 25:8843-8853 (2005)]. Theimmunosuppressant drug FK506 (tacrolimus) attenuated microglialactivation and extended the life span of P301S transgenic mouse model ofFTD [Yoshiyama et al., Neuron 53:337-351 (2007)]. Further, a growingnumber of studies suggest that proinflammatory cytokines, such asinterleukin-1 (IL-1), interleukin-6 (IL-6), and nitric oxide releasedfrom astrocytes can accelerate MAPT pathology and formation ofneurofibrillary tangles (NFTs) in vitro [Li et al., J Neurosci,23:1605-1611 (2003); Quintanilla et al., Exp Cell Res, 295:245-257(2004); Saez et al., In Vivo, 18:275-280 (2004)].

The toll-like receptors (TLRs) are a group of transmembrane receptorswhose cytoplasmic portions are highly similar, having a high similarityto the interleukin-1 (IL-1) receptor. That cytoplasmic portion is nowreferred to as the Toll/IL-1 receptor (TIR) domain. The extracellularportions are structurally unrelated. The TLRs recognize pathogencomponents. [Takeda et al., Seminars in Immunology, 16:3-9 (2004).]

TLR4 plays a fundamental role in pathogen recognition in recognizinglipopolysaccharide (LPS) found in most gram-negative bacteria as well asother molecules. This receptor also plays a role in activation of innateimmunity. TLR4 pathway activation can be an indicator of an infection.

TLR4 typically associates with the adapter molecule, MD2, CD14 and thelipopolysaccharide binding molecule (LPB) when associating with LPS.Signaling occurs through a series of cytoplasmic molecules in what arereferred to as the myeloid differentiation factor 88- (MyD88-) dependentpathway common to all TLRs, and the MyD88-independent pathway shared byTLR3 and TLR4. TLR3 recognizes double-stranded RNA and its activationoccurs under different conditions from TLR4 activation.

Signaling induced by LPS via the MyD88-independent pathway leads toactivation of the transcription factor IRF-3, and thereby induces IFN-β.IFN-β, in turn, activates Stat1, leading to the induction of severalIFN-inducible genes. LPS-induced activation of NF-κB and JNK appears tobe independent of the presence of MyD88. [Takeda et al., Seminars inImmunology, 16:3-9 (2004).]

TLR4 is present in cells of the immune system such as B cells, T cellsand macrophages, as well as cells of the CNS. TLR4 is an importantmediator of the innate immune response, and significantly contributes toneuroinflammation induced by brain injury. The TLR4-mediatedneuroinflammation typically proceeds through the above TLR4/adapterprotein MyD88 signaling pathway.

Mao et al., J Neurotrauma, May 14 (2012) reported the potentialneuroprotective mechanisms of pituitary adenylate cyclase-activatingpolypeptide-(PACAP-) pretreatment in a rat model of traumatic braininjury (TBI). It was found that TBI induced significant upregulation ofTLR4 with peak expression occurring 24 hours post-trauma.

Pretreatment with PACAP significantly improved motor and cognitivedysfunction, attenuated neuronal apoptosis, and decreased brain edema.That pretreatment inhibited TLR4 upregulation as well as that of itsdownstream signaling molecules, MyD88, p-IκB, and NF-κB, and suppressedincreases in levels of the downstream inflammatory agents,interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), in the braintissue around the injured cortex and in the hippocampus. PACAP treatmentthus exerted a neuroprotective effect in this rat model of TBI,potentially via inhibiting a secondary inflammatory response mediated bythe TLR4/MyD88/NF-κB signaling pathway in microglia and neurons, therebyreducing neuronal death and improving the outcome following TBI.

Traumatic brain injury (TBI) is a “signature” injury of recent militaryconflicts and is associated with psychiatric symptoms and long-termcognitive disability. Chronic traumatic encephalopathy (CTE), ahyperphosphorylated tau protein-linked neurodegenerative disorder(tauopathy) reported in athletes with multiple concussions, sharesclinical features with TBI in military personnel exposed to explosiveblast. CTE also shares pathology found in boxers that was previouslyknown as dementia pugilistica. [Gandy et al., Sci. Transl. Med.4:1341-1343 (May 12, 2012).]

Goldstein et al., Sci. Transl. Med. 4:134ra60 (2012), investigated theconnection between TBI and CTE in a series of postmortem brains fromU.S. military veterans with blast exposure and/or concussive injury.Those authors reported evidence for CTE neuropathology in the militaryveteran brains that is similar to that observed in the brains of youngamateur American football players and a professional wrestler. Theinvestigators developed a mouse model of blast neurotrauma that mimicstypical blast conditions associated with military blast injury anddiscovered that blast-exposed mice also demonstrate CTE neuropathology,including tau protein hyperphosphorylation, myelinated axonopathy,microvascular damage, chronic neuroinflammation, and neurodegeneration.

The mouse neuropathology was reported to be accompanied by functionaldeficits, including slowed axonal conduction, reduced activity-dependentlong-term synaptic plasticity, and impaired spatial learning and memorythat persisted for 1 month after exposure to a single blast. Theinvestigators then showed that blast-induced learning and memorydeficits in the mice were reduced by immobilizing the head during blastexposure.

Neuropathological findings in the military veterans with blast exposureand/or concussive injury and young-adult athletes with repetitiveconcussive injury were consistent with those authors' previous CTE casestudies [McKee et al., J Neuropathol Exp Neurol 68:709-735 (2009); McKeeet al., J Neuropathol Exp Neurol 69:918-929 (2010)], and were reportedto be readily differentiated from neuropathology associated withAlzheimer's disease, frontotemporal dementia, and other age-relatedneurodegenerative disorders.

Apolipoprotein E (ApoE) is a class of apolipoprotein found in thechylomicron and intermediate-density lipoprotein (IDLs) that binds to aspecific receptor on liver cells and peripheral cells. ApoE has beenstudied for its role in several biological processes not directlyrelated to lipoprotein transport, its more-studied function, includingAlzheimer's disease, immunoregulation, and cognition.

ApoE is 299 amino acids long and transports lipoproteins, fat-solublevitamins, and cholesterol into the lymph system and then into the blood.It is synthesized principally in the liver, but has also been found inother tissues such as the brain. In the nervous system, non-neuronalcell types, most notably astroglia and microglia, are the primaryproducers of ApoE, whereas neurons preferentially express the receptorsfor ApoE.

There are seven currently identified mammalian receptors for ApoE thatbelong to the evolutionarily conserved low density lipoprotein receptorgene family. ApoE is a polymorphic gene with three major isoforms,ApoE2, ApoE3, ApoE4, which translate from three alleles of the gene, ofwhich ApoE-ε3 is the “normal” allele, and ApoE-E2 and ApoE-24 aredysfunctional alleles.

ApoE4 has been implicated in atherosclerosis and Alzheimer's disease,impaired cognitive function, and reduced neurite outgrowth. The ApoE4variant is the largest known genetic risk factor for late-onset sporadicAlzheimer's Disease (AD) in a variety of ethnic groups. Caucasian andJapanese carriers of two E4 alleles have between 10 and 30 times therisk of developing AD by 75 years of age, as compared to those notcarrying any E4 alleles.

Although 40-65% of AD patients have at least one copy of the 4 allele,ApoE4 is not a determinant of the disease. At least one-third ofpatients with AD are ApoE4 negative and some ApoE4 homozygotes neverdevelop the disease. However, those with two E4 alleles have up to 20times the risk of developing AD.

In addition, ApoE4 overexpression in mouse neurons resulted inhyperphosphorylation of tau and the development of motor problems,accompanied by muscle wasting, loss of body weight and premature death.[Tesseur et al., Am J Pathol, 156(3):951-964 (2000).] On the other hand,treatment of neurons with exogenously supplied ApoE isoforms (E2 or E4)affects several downstream signaling cascades in neurons: decreased taukinase phosphorylation and inhibition of tau phosphorylation at Thr171and Ser202/Thr205 epitopes in the primary neuronal culture. ApoE canalter levels of tau kinases and phospho-tau epitopes, potentiallyaffecting tau neuropathological changes seen in AD brains. [Hoe et al.,Molecular Degeneration, 1:8 (2006).]

Eisenberg and co-workers have studied the formation of beta-sheetfibrils from self-aggregating tau protein, and found that a particularhexapeptide can inhibit their formation by interfering with the ‘stericzipper’ of the beta-sheet fibril. However, the inhibiting peptide is toolarge to penetrate deeply into the brain nor does it appear to penetratethe brain cells in which the tau fibrils form. See, Sawaya et al.,Nature, 447:453-457 (2007); Landau et al., PLoS Biology, 9 (6):e1001080(2011); and Sievers et al., Nature, DOI:10.1038/nature10154 (2011). Itwould therefore be beneficial if an inhibitor of the formation oftau-containing NFTs could be found that penetrates the brain and otherCNS structures, as well as the cells of those structures.

Alzheimer's disease (AD) poses a huge unmet medical need, with anestimated 35 million current patients worldwide and no disease-modifyingtreatment available. The two classes of drugs currently used for AD,cholinesterase inhibitors and memantine, only transiently enhancecognitive function in these patients.

The causative agent in AD pathology is generally accepted to beamyloid-β (Aβ), Aβ₄₂. Aβ is a 39-42-residue proteolysis product ofamyloid precursor protein (APP) that is an integral membrane proteinexpressed in many tissues and concentrated in the synapses of neurons.

Transgenic animals with increased levels of Aβ can model AD, and Aβlevels in postmortem AD brains are correlated with the degree ofcognitive impairment and neuropathology [Tanzi et al., Cell 120:545-555(2005)]. This correlation is higher for soluble Aβ than for Aβ-richplaques, implicating soluble Aβ in AD pathogenesis [Naslund et al., J AmMed Assoc, 283:1571-1577 (2000).]

It is believed that the critical pathogenic role of soluble Aβ is toxicsignaling via the α-7 nicotinic acetylcholine receptor (α7nAChR), asdemonstrated a decade ago. Aβ binds this receptor with high affinity[Wang et al., J Biol Chem 275:5626-5632 (2000); Wang et al., J Neurochem75:1155-1161 (2000)], activating ERK2, which phosphorylates the tauprotein [Wang et al., J Biol Chem 278:1547-31553 (2003)]. ERK2 is alsoknown as mitogen-activated protein kinase 1 (MAPK1) noted earlier.

Persistent abnormal hyperphosphorylation of tau proteins results inneurofibrillary tangles (NFTs), a prominent neuropathological feature inAD brain, and the magnitude of these lesions correlates with theseverity of AD symptoms [Delacourte et al., Neurology 52:158-1165(1999); Delacourte et al., Neurology 43:93-204 (1998).] The NFTs areinitially intracellular, and become extracellular ghost tangles afterdeath of the neuron [Mandelkow et al., Trends in Cell Biology, 8:425-427(1998)].

Aβ peptide has been shown to induce tau phosphorylation in several invitro experimental systems [Johnson et al., J Alzheimers Dis 1:29-351(1999)], and Aβ-induced tau phosphorylation has been demonstrated to bedependent on α7nAChR, because pretreatment of tissues with α7nAChRantagonists or with Aβ₁₂₋₂₈, which inhibit the Aβ₄₂-α7nAChR interaction,reduces Aβ₄₂-induced tau phosphorylation [Wang et al., J Biol Chem278:547-31553 (2003)].

Phosphorylation of some sites appears to regulate microtubule-bindingproperties (e.g., Ser262 and Ser356) [Mandelkow et al., Trends in CellBiology, 8:425-427 (1998)]. On the other hand, phosphorylation at one ormore of 202Ser, 231Thr and 181Thr is found in tau-containing NFTs [Wanget al., J Biol Chem 278:31547-31553 (2003); Wang et al., Biol Psychiatry67:522-530 (2010)].

The critical role of the α7nAChR in mediating neurofibrillary pathologyis further supported by at least two findings: 1) protracted incubationof Aβ₄₂ with SK-N-MC cells that over-express α7nAChRs promotes NFTs, and2) antisense-α7nAChR oligonucleotides that reduce α7nAChR levels abolishAβ₄₂-induced neurofibrillary lesions [Wang et al., J Biol Chem278:31547-31553 (2003)]. These data suggest that chronic perturbation ofthe α7nAChRs with Aβ₄₂ in AD brains leads to neurofibrillaryphosphorylated tau-containing lesions.

As discussed in detail hereinafter, the present invention provides amethod to inhibit Aβ₄₂-induced hyperphosphorylation of tau proteins byinhibiting one or more signaling pathways that utilize the signalingscaffold, filamin A (FLNA). In one pathway, Aβ and α7nAChR interactleading to the recruitment of FLNA. In another pathway, TLR4 isactivated by Aβ₄₂ or its cognate ligand, LPS for example, and theTLR4-mediated signaling is activated through the recruitment of FLNA tothe TLR4 receptor. Aβ₄₂ induces FLNA recruitment to α7nAChR or TLR4 aswell as tau phosphorylation can be observed by incubating 250,000 cellsin 250 μl of oxygenated Kreb's-Ringer with 1 nM Aβ₄₂. This Aβ₄₂-mediatedeffect was found to be plateaued at 100 nM.

The treatment approach disclosed below is targeted at inhibitinghyperphosphorylation of tau proteins mediated by FLNA using a compoundthat binds FLNA with high affinity. This binding is believed to alterthe conformation of FLNA and prevent it from interacting with othersignaling molecules such as α7nAChRs, thereby inhibiting thehyperphosphorylation of the tau protein.

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates a method of inhibitinghyperphosphorylation [phosphorylation at one or more of serine-202 (also202Ser and S²⁰²), threonine-231 (also 231Thr and T²³¹) and threonine-181(also 181Thr and T¹⁸¹) in addition to phosphorylation that may bepresent at any other site] of the tau protein that comprises the stepsof administering to central nervous system cells in recognized(diagnosed) need thereof an effective amount of a compound or apharmaceutically acceptable salt thereof that binds to filamin A (FLNA)or binds to a pentapeptide of filamin A of SEQ ID NO: 1 as described inExample 1, e.g., inhibits at least about 60 percent and more preferablyabout 70 percent of the FITC-labeled naloxone binding when present at a10 μM concentration and using unlabeled naloxone as the controlinhibitor at the same concentration. The compound is preferably ofSeries A, B, C-1, C-2, D or E as described hereinafter, and preferablycontains at least four of the six pharmacophores of FIGS. 35-40. Theadministration is carried out in the absence of a mu opioid receptor(MOR)-binding effective amount of a separate MOR agonist or antagonistmolecule.

The use of a single stereoisomer or mixture of stereoisomers, or apharmaceutically acceptable salt of a contemplated compound is alsocontemplated. The contemplated administration can take place in vivo orin vitro, and is typically repeated over a period of days or months whenadministered in vivo.

Another aspect of the invention contemplates a method of inhibiting aTLR4-mediated immune response such as inflammation of cells of the CNS.A contemplated method comprises administering to TLR4-containing cellsin recognized (diagnosed) need thereof an effective amount of a compoundor a pharmaceutically acceptable salt thereof that binds to filamin A orbinds to a pentapeptide of filamin A (FLNA) of SEQ ID NO: 1 as describedin Example 1, inhibits at least about 60 percent and more preferablyabout 70 percent of the FITC-labeled naloxone binding when present at a10 μM concentration and using unlabeled naloxone as the controlinhibitor at the same concentration. A contemplated compound ispreferably of Series A, B, C-1, C-2, D or E as described herein, andpreferably contains at least four of the six pharmacophores of FIGS.35-40. The administration is preferably carried out in the absence of amu opioid receptor (MOR)-binding effective amount of a separate MORagonist or antagonist molecule.

The use of a single stereoisomer or mixture of stereoisomers, or apharmaceutically acceptable salt of a contemplated compound is alsocontemplated. The contemplated administration can take place in vivo orin vitro, and is typically repeated over a period of days or months whenadministered in vivo to the cells of a host animal such as a human.

In one aspect of an above method, tau hyperphosphorylation of one ormore of S²⁰², T²³¹ and T¹⁸¹ occurs through the interaction of Aβ andα7nAChR via the scaffolding protein filamin A (FLNA). In another aspectof a contemplated method, such tau hyperphosphorylation occurs via aTLR4-mediated immune response in a presently unknown mechanism that alsoinvolves the intermediacy of FNLA.

It is presently believed that each of the above pathways, Aβ-α7nAChR andTLR4, can operate at the same time and also independently. IllustrativeCNS conditions that exhibit one or both of Aβ-α7nAChR-mediated and/orTLR4-mediated tau phosphorylations of one or more of S²⁰², T²³¹ and T¹⁸¹include those of persons and other animals whose CNS cells exhibit animmune response such as inflammation induced by brain injury such astraumatic brain injury (e.g., concussion), chronic traumaticencephalopathy, those having Alzheimer's disease (AD) symptoms,frontotemporal dementia (FTD), progressive supranuclear palsy, dementiapugilistica and corticobasal degeneration and also infection by one orboth of Gram positive and Gram negative bacteria.

The binding inhibition to a SEQ ID NO: 1 pentapeptide by a contemplatedcompound is determined as discussed in Example 1. A contemplatedcompound is substantially free from binding with any other portion ofFLNA at the concentration of contemplated compound used. Substantialfreedom from binding with any other portion of FLNA can be determinedusing a titration assay such as that shown in FIG. 48A herein [FIG. 3 ofWang et al., PLoS One. 3 (2):e1554 (2008)], which in that figureindicates the presence of two binding site regions by the two inflectionpoints shown in the plot, whereas the presence of a single binding siteis indicated by the presence of a single inflection point in such a plot(FIG. 48D). Substantial freedom from binding with any other portion ofFLNA can also be inferred from functional data such as a cytokinerelease assay illustrated hereinafter that indicate contemplatedcompounds do not bind the second site on FLNA because the compounds areeffective over a wide range of concentrations, unlike those compoundssuch as naloxone and naltrexone that bind to two binding sites on FLNA.

In presently preferred embodiments, the present invention contemplates amethod of inhibiting phosphorylation of the tau protein at one or moreof S²⁰², T²³¹ and T¹⁸¹ that comprises the step of administering to cellsof the central nervous system in recognized (diagnosed) need such asbrain cells an effective amount of a compound of one or more of SeriesA, Series B, Series C-1 and Series C-2, Series D and Series E, astereoiosomer or a pharmaceutically acceptable salt thereof. The cells,in vivo or in vitro, such as brain cells in recognized need (diagnosed)are cells in those tissues or organs of a mammalian subject that exhibitan immune response such as inflammation induced by brain injury liketraumatic brain injury, chronic traumatic encephalopathy, those havingAlzheimer's disease (AD) symptoms, frontotemporal dementia (FTD),progressive supranuclear palsy, dementia pugilistica and corticobasaldegeneration and also infection by one or both of Gram positive and Gramnegative bacteria.

The administration is preferably carried out in the absence of aMOR-binding effective amount of a separate MOR agonist or antagonistmolecule, and is often carried out a plurality of times over a period ofdays or months.

Also contemplated is the use of a compound or a pharmaceuticallyacceptable salt thereof in the manufacture of a medicament forinhibiting phosphorylation of the tau protein in cells of the centralnervous system that are in recognized need of treatment. Such a compoundor its salt binds to a pentapeptide of filamin A of SEQ ID NO: 1,inhibits at least about 60 percent and more preferably about 70 percentof the FITC-labeled naloxone binding when present at a 10 μMconcentration and using unlabeled naloxone as the control inhibitor atthe same concentration. This use contemplates manufacture of amedicament that inhibits tau hyperphosphorylation (phosphorylation)through the interaction of Aβ and α7nAChR via the scaffolding proteinfilamin A (FLNA), as well as such tau hyperphosphorylation that occursvia a TLR4-mediated immune response that is also believed to involve theintermediacy of FNLA as are noted above.

The general structures of the compounds of each series are shown below,followed by more specific disclosures for the various letters andR-groups.

Individual optical isomers and mixtures of optical isomers of thosecompounds of the above Formulas are also contemplated, as arepharmaceutically acceptable salts of those compounds.

A contemplated compound described above or its pharmaceuticallyacceptable salt is typically administered in an effective amountdissolved or dispersed in a pharmaceutical composition. Thatpharmaceutical composition can be in solid or liquid form.

The invention also contemplates a method of:

a) inhibiting α7nAChR-FLNA association (Complex formation);

b) inhibiting Aβ-induced α7nAChR-mediated signaling of extracellularsignal-regulated kinase 2 [ERK2; also known as mitogen-activated proteinkinase 1 (MAPK1)];

c) inhibiting Aβ₄₂-induced association of TLR4 with FLNA;

d) inhibiting Aβ₄₂-induced impairment in α7nAChR function, e.g., calciuminflux, after α7nAChR stimulation;

e) restoring Aβ₄₂-induced impairment in N-methyl-D-aspartate (NMDA)receptor (NMDAR) function, e.g., calcium influx after stimulation ofNMDAR with co-agonists NMDA and glycine;

f) restoring the Aβ₄₂-induced impairment in insulin receptor (IR)function as measured by one or both of phosphorylation of the subunitIRβ and association with the IRS-1 signaling molecule;

g) inhibiting Aβ₄₂-induced impairment of K⁺-evoked cellular calciuminflux;

h) reducing the formation of tau-containing NFTs and also Aβ₄₂aggregates (neuritic plaques) in the presence of Aβ₄₂; and

i) inhibiting Aβ₄₂-induced inflammatory cytokine production. Each ofthose methods is carried out by administering to cells of the centralnervous system such as brain cells an effective amount of abefore-described compound that binds to the FLNA pentapeptide of SEQ IDNO: 1. The administration is carried out in the absence of a MOR-bindingeffective amount of a separate MOR agonist or antagonist molecule.

A still further aspect of this invention contemplates a method ofpromoting cartilage repair in a mammal having osteoarthritis, such asthat induced by collagenase and/or surgery or other causes. This methodcomprises the steps of administering to multipotent mesenchymal stemcells in a mammal in recognized need (diagnosed) thereof an effectiveamount of a compound or a pharmaceutically acceptable salt thereof thatbinds to filamin A or binds to a pentapeptide of filamin A (FLNA) of SEQID NO: 1 as described in Example 1, e.g., inhibits at least about 60percent and more preferably about 70 percent of the FITC-labelednaloxone binding when present at a 10 μM concentration and usingunlabeled naloxone as the control inhibitor at the same concentration.The compound is preferably of Series A, B, C-1, C-2, D or E as describedherein, and preferably contains at least four of the six pharmacophoresof FIGS. 35-40. The administration is carried out in the absence of a muopioid receptor (MOR)-binding effective amount of a separate MOR agonistor antagonist molecule.

The use of a single stereoisomer or mixture of stereoisomers, or apharmaceutically acceptable salt of a contemplated stereoisometriccompound is also contemplated. The contemplated administration can takeplace in vivo or in vitro, and is typically repeated when administeredin vivo.

The present invention has several benefits and advantages.

One benefit is that a contemplated method inhibits Aβ signaling throughα7nAChR that is believed superior to targeting the receptor itself.Disabling the Aβ-induced α7nAChR signaling without directly affectingthe α7nAChRs avoids altering the sensitivity or cell surface level ofthe receptors, an insidious problem with using chronic receptor agonistsor antagonists.

An advantage of this invention is that this approach appears toselectively affect the robust increase in filamin recruitment by Aβwhile preserving basal coupling, suggesting that the compounds used inthe method reduce the pathological signaling by Aβ, while retainingphysiological α7nAChR signaling.

Another benefit of the invention is that administration of acontemplated compound inhibits the in vitro and in vivo phosphorylationof the tau protein.

Another advantage of the invention is that when a contemplated compoundis administered in vivo, the administration inhibits the formation ofNFTs in the brain of a subject mammal to which a contemplated compoundis administered.

Yet another benefit of the invention is that administration of acontemplated compound can provide the benefits of one or more of themethods enumerated above by binding of that compound to the FLNApentapeptide of SEQ ID NO: 1 disrupting one or more of the newly-foundinteractions of FLNA.

Yet another advantage of the invention is that its use can lessen theeffects of tau phosphorylation in persons or other animals with headinjuries and resultant TLR4-mediated inflammation.

Still further benefits and advantages will be apparent to those skilledin the art from the disclosures that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a part of this disclosure,

FIG. 1 shows a heightened α7nAChR-FLNA association in frontal cortex ofAD transgenic mice and AD patients. Frontal cortical synaptosomes from6-month-old AD transgenic/wild-type mice (n=4) and 4 matching AD/controlhuman pairs were analyzed for their baseline α7nAChR-FLNA complexcontents. The α7nAChR-FLNA complexes in the solubilized frontal corticalsynaptosomes were immunoprecipitated with anti-FLNA and the α7nAChRlevels in anti-FLNA immunoprecipitate were determined by Westernblotting with anti-α7nAChR antibodies. Blots were then stripped andre-probed with anti-FLNA as a loading control. Densitometric scanningwas used for quantification.

FIG. 2 in two panels as FIG. 2A and FIG. 2B, illustrates high-affinityFLNA-binding compounds reduce α7nAChR-FLNA association. Frontal corticalsynaptosomes from 2-month-old rats (n=4) were treated with 0.1 or 1 nMconcentrations of compounds [A0033, A0040, A0053, A0068, B0055, C0105M,C0114M, C0137M and C0138M] either simultaneously (Sim) with or 10minutes prior (10′ pr) to Aβ₄₂ and were analyzed for their α7nAChR-FLNAcomplex contents. The α7nAChR-FLNA complexes in the solubilizedsynaptosomes were immunoprecipitated with immobilized anti-FLNA and theα7nAChR and FLNA levels in the anti-FLNA immunoprecipitates determinedby Western blotting (FIG. 2A) and quantified by densitometry (FIG. 2B).n=3. Data are means±SEM. **p<0.05, *p<0.01 vs. Aβ₄₂ alone. The letterdesignation “M” that accompanies many of the “C-series” compounds isomitted from FIG. 2, the remaining figures and most discussions of thefigures and compounds hereinafter for ease in expression. Structuralformulas of the compounds used in this and the other figures areprovided hereinafter.

FIG. 3 in two panels as FIG. 3A and FIG. 3B, illustrates thatFLNA-binding compounds reduce Aβ-induced α7nAChR-mediated ERK2signaling. In the same treated synaptosomes used in FIG. 2, levels ofphosphorylated (activated) ERK2 were measured in immunoprecipitates ofERK2. Aβ₄₂ strongly activated extracellular signal-regulated kinase 2[ERK2; also known as mitogen-activated protein kinase 1 (MAPK1)], andall compounds studied reduced this activation with 10-minutes ofpretreatment. Compound C0105 also reduced pERK2 with simultaneousadministration. Immunoprecipitates were determined by Western blotting(FIG. 3A) and quantified for four compounds by densitometry (FIG. 3B).n=3. Data are means±SEM. **p<0.05, *p<0.01 vs. Aβ₄₂ alone.

FIG. 4 in four panels as FIGS. 4A-4D, illustrates that FLNA-bindingcompounds reduce tau phosphorylation at all three phosphorylation sites.In the same treated synaptosomes used in FIGS. 2 and 3, levels of tauprotein phosphorylated at S²⁰², T²³¹ and T¹⁸¹ were measured inimmunoprecipitates using an anti-tau antibody that does not distinguishits phosphorylation state. The three phosphoepitopes of tau weredetected in immunoprecipitates using specific antibodies. Aβ₄₂ stronglypromotes tau phosphorylation at all three sites, and all compoundsreduced this phosphorylation with 10-minute pretreatment. Compound C0105also reduced tau phosphorylation with simultaneous administration.Immunoprecipitates were determined by Western blotting (FIGS. 4A-4C) andquantified by densitometry (FIG. 4D). Data are means±SEM. **p<0.05,*p<0.01 vs. Aβ₄₂ alone.

FIG. 5 in three panels illustrates that compounds C0105 and C0114 reduceAβ₄₂-induced increases in association of FLNA with α7nAChR and TLR4.Synaptosomes were prepared from organotypic FCX slice cultures treatedwith 0.1 or 1 nM concentrations of compounds simultaneously with Aβ₄₂and analyzed for their α7nAChR-FLNA complex contents. The extent of FLNAassociation with α7nAChR, TLR4, IR and MOR was assessed in thesolubilized synaptosomes by immunoprecipitating with immobilizedanti-FLNA and Western blot detection (FIG. 5A) using antibodies specificto each receptor. Blots were analyzed by densitometric quantitation(FIG. 5B). Although Aβ₄₂ greatly increased association of α7nAChR andTLR4 with FLNA, levels of IR and MOR association with FLNA wereunchanged. C0105 and C0114 decreased these Aβ₄₂-induced increases.Percent inhibition is depicted in FIG. 5C. n=3. Data are means±SEM.**p<0.05, *p<0.01 vs. Aβ₄₂ alone; #p<0.01 vs. vehicle.

FIG. 6 in three panels illustrates that contacting the synaptosomes withcompounds C0105 (0.1, 1 and 10 nM) and C0114 (1 and 10 nM) reduced tauphosphorylation at all three phosphorylation sites. In the same treatedsynaptosomes used in FIG. 5, levels of tau protein phosphorylated atS²⁰², T²³¹ and T¹⁸¹ were measured in immunoprecipitates using ananti-tau antibody that does not distinguish its phosphorylation state.The three phosphoepitopes of tau were detected in immunoprecipitatesusing specific antibodies. Aβ₄₂ strongly promotes tau phosphorylation atall three sites, and both compounds significantly reduced thisphosphorylation. Western blots (FIG. 6A) were analyzed by densitometricquantitation (FIG. 6B). Percent inhibition is depicted in FIG. 6C. n=3.Data are means±SEM. **p<0.05, *p<0.01 vs. Aβ₄₂ alone; #p<0.001,##p<0.007, ###p<0.003 vs. Aβ₄₂-free vehicle group.

FIG. 7 illustrates that each of Compounds C0105 and C0114 restoresAβ₄₂-induced impairment in α7nAChR function. In the same synaptosomesfrom treated FCX cultures, Aβ₄₂ significantly impairs calcium influxafter stimulating with PNU282987, a full agonist of α7nAChR, andCompounds C0105 and C0114 each prevent this impairment. n=3. Data aremeans±SEM. *p<0.01 vs. vehicle; #p<0.01 vs. Aβ₄₂ alone.

FIG. 8 illustrates that each of Compounds C0105 and C0114 restoresAβ₄₂-induced impairment in NMDAR function. Aβ₄₂ significantly impairscalcium influx after stimulating with NMDA and glycine, co-agonists ofNMDAR, and Compounds C0105 and C0114 prevent this impairment. n=3. Dataare means±SEM. *p<0.01 vs. vehicle; ##p<0.05, #p<0.01 vs. Aβ₄₂ alone.

FIG. 9 in two panels illustrates that each of compounds C0105 and C0114restores levels of NMDAR-associated signaling molecules. Signalingfunction of NMDAR was also assessed by measuring levels of six differentsignaling molecules co-immunoprecipitating with NR-1, the obligatorysubunit of NMDAR, after co-stimulation with glycine and NMDA (FIG. 9A).Aβ₄₂ suppressed levels of association of all six signaling componentswith NR-1 confirming the NMDAR dysfunction illustrated in FIG. 8 (FIG.9B). n=3. Data are means±SEM. **p<0.05, *p<0.01 vs. Aβ₄₂ alone; #p<0.01vs. vehicle.

FIG. 10 in two panels C0105 and C0114 illustrates that each of CompoundsC0105 and C0114 restores impairment in IR function induced by Aβ₄₂. Aβ₄₂impaired signaling of IR as measured by phosphorylation of IRβ and itsassociation with the signaling molecule IRS-1 as shown by western blots(FIG. 10A) and densitometric measurements (FIG. 10B). *p<0.01 vs. Aβ₄₂alone; #p<0.01 vs. vehicle.

FIG. 11 illustrates that each of compounds C0105 and C0114 can reducecell death as indicated by reduced K⁺-evoked calcium influx. Aβ₄₂reduced K⁺-evoked Ca⁺² influx, indicating dying or non-functional cells.Contacting the cells with either of compounds C0105 and C0114 preventthat Aβ₄₂-induced impairment. n=3. Data are means±SEM. **p<0.05, *p<0.01vs. vehicle; #p<0.01 vs. Aβ₄₂ alone.

FIG. 12 in two sets of three panels illustrates that administration ofcompound C0105 to frontocortical brain slice cultures preventsAβ₄₂-induced NFTs. Incubation with Aβ₄₂ produced NFTs as visualized byphosphorylated tau (pTau) immunoreactivity (FIG. 12A). Co-incubation ofAβ₄₂ with compound C0105 prevented this neuropathology (FIG. 12B).Vehicle-treated slices are depicted in FIG. 12C. Lower panels are highermagnification.

FIG. 13 in two sets of three panels illustrates that administration ofcompound C0105 to frontocortical brain slice cultures dramaticallyreduces immunostaining of Aβ₄₂ aggregates. Incubation with Aβ₄₂ producedamyloid deposits as visualized by Aβ₄₂ immunoreactivity (FIG. 13A), andco-incubation with Aβ₄₂ and Compound C0105 prevented this neuropathology(FIG. 13B). Vehicle treated slices are depicted in FIG. 13C. Lowerpanels are higher magnification.

FIG. 14 in three parts illustrates that the systemic administration ofCompound C0105 to mice decreased Aβ₄₂-induced FLNA association with bothα7nAChR and toll-like receptor 4 (TLR4). Thus, synaptosomes preparedfrom prefrontal cortex and hippocampus of mice receiving continuousintracerebroventricular (ICV) infusion of Aβ₄₂ or vehicle and twicedaily injections of Compound C0105 or vehicle were analyzed for theirFLNA-α7nAChR/TLR4 interactions. The extent of FLNA association withα7nAChR or TLR4 was assessed in the solubilized synaptosomes byimmunoprecipitation with immobilized anti-FLNA and Western blotdetection (FIG. 14A) using antibodies specific to each receptor.Numerals outside of and to the left of the blots are molecular weightpositions within the blots. Blots were analyzed by densitometricquantitation (FIG. 14B). Aβ₄₂ greatly increased association of α7nAChRand TLR4 with FLNA, and Compound C0105 decreased these Aβ₄₂-inducedincreases. Percent inhibition is depicted in FIG. 14C. n=3. Data aremeans±SEM. *p<0.01 vs. sham, vehicle; #p<0.01 vs. Aβ₄₂, vehicle.

FIG. 15 in three parts illustrates that administration of Compound C0105to mice reduces tau phosphorylation at all three phosphorylation sites.In the same treated synaptosomes used in FIG. 14, levels of tau proteinphosphorylated at S²⁰², T²³¹ and T¹⁸¹ were measured inimmunoprecipitates using an anti-tau antibody that does not distinguishits phosphorylation state. The three phosphoepitopes of tau weredetected in immunoprecipitates using specific antibodies. Aβ₄₂ stronglypromotes tau phosphorylation at all three sites, and both compoundssignificantly reduced this phosphorylation. Western blots (FIG. 15A)were analyzed by densitometric quantitation (FIG. 15B). Numerals outsideof and to the left of the blots of FIG. 15A are molecular weightpositions within the blots. Percent inhibition is depicted in FIG. 15C.Data are means±SEM. *p<0.01 vs. sham, vehicle; #p<0.01 vs. Aβ₄₂,vehicle.

FIG. 16 in three parts illustrates that administration of Compound C0105to mice reduces Aβ₄₂-α7nAChR complexes. Twice daily treatment of micewith Compound C0105 greatly reduced the level of Aβ₄₂-α7nAChR complexesin both prefrontal cortex and hippocampus, n=7 or n=8. Western blots(FIG. 16A) were analyzed by densitometric quantitation (FIG. 16B).Numerals outside of and to the left of the blots of FIG. 16A aremolecular weight positions within the blots. Percent inhibition isdepicted in FIG. 16C. Data are means±SEM. *p<0.01 vs. sham, vehicle;#p<0.01 vs. Aβ₄₂, vehicle.

FIG. 17 illustrates that Compound C0105 treatment reduces Aβ₄₂-inducedα7nAChR dysfunction. Twice daily treatment of mice with Compound C0105normalized the Aβ₄₂-induced impairment in calcium influx followingstimulation with the full α7nAChR agonist PNU282987. n=7 or n=8. Dataare means±SEM. *p<0.01 vs. sham, vehicle; #p<0.01 vs. Aβ₄₂, vehicle;+p<0.01 vs. vehicle- and Compound C0105-treated sham groups.

FIG. 18 illustrates that Compound C0105 treatment of mice reducesAβ₄₂-induced NMDAR dysfunction. Aβ₄₂ significantly impairs calciuminflux after stimulating with NMDA and glycine, co-agonists of NMDAR,and Compound C0105 prevents this impairment. n=7 or n=8. Data aremeans±SEM. *p<0.01 vs. sham, vehicle; #p<0.01 vs. Aβ₄₂, vehicle; +p<0.01vs. vehicle- and Compound C0105-treated sham groups.

FIG. 19 illustrates that Compound C0105 treatment of mice reduces celldeath as measured by K⁺-evoked Ca⁺² influx. Aβ₄₂ reduced K⁺-evoked Ca⁺²influx, indicating dying or non-functional cells. Compound C0105 reducesthis Aβ₄₂-induced impairment. n=7 or n=8. Data are means±SEM. *p<0.01vs. sham, vehicle; #p<0.01 vs. Aβ₄₂, vehicle; ++p<0.05 vs. vehicle- andCompound C0105-treated sham groups.

FIG. 20 in two panels illustrates that Compound C0105 treatment of micenormalizes Aβ₄₂-induced NMDAR signaling impairments. Signaling functionof NMDAR was also assessed by measuring levels of six differentsignaling molecules (PLCγ, nNOS, pY⁴⁰²PyK2, PSD-95, PKCγ, pY⁴¹⁶Src, andNR1) co-immunoprecipitating with NR-1, the obligatory subunit of NMDAR,after co-stimulation with glycine and NMDA (FIG. 20A). Numerals outsideof and to the left of the blots are as discussed before. Aβ₄₂ suppressedlevels of association of all six signaling components with NR-1confirming the NMDAR dysfunction illustrated in FIG. 18 (FIG. 20B). n=7or n=8. Data are means±SEM. +p<0.01 vs. basal level in control vehiclegroup; *p<0.01 vs. NMDA/glycine-stimulated level in sham, vehicle group;#p<0.01 vs. NMDA/glycine-stimulated level in ICV Aβ₄₂, vehicle.

FIG. 21 in two panels illustrates that Compound C0105 treatment of micenormalizes Aβ₄₂-induced insulin receptor signaling impairments. Aβ₄₂impaired signaling of IR as measured by phosphorylation of IRβ and itsassociation with the signaling molecule IRS-1. Immunoprecipitatesprepared using immobilized anti-IRβ are shown in FIG. 21A, whereinnumerals outside of and to the left of the blots are as discussedbefore. FIG. 21B shows results obtained after removal of the antibodies,transfer to nitrocellulose membranes and Western blotting withantibodies to the species noted on the ordinates, followed byquantitation using densitometry. Compound C0105 normalized theseimpairments. n=7 or n=8. Data are means±SEM. *p<0.01 vs.insulin-stimulated level in sham, vehicle group; #p<0.01 vs.insulin-stimulated level in ICV Aβ₄₂, vehicle group.

FIG. 22 illustrates that Compound C0105 treatment of mice nearlyabolishes Aβ₄₂-induced cytokine production. Aβ₄₂ increased levels ofcytokines IL-6, TNFα and IL-1β measured by a fluorescence ELISA assayusing FITC. Compound C0105 almost abolished the production of these 3cytokines. n=7 or n=8. Data are means±SEM. *p<0.01 vs. respectivecytokine level in sham, vehicle group; #p<0.01 vs. respective cytokinelevel in ICV Aβ₄₂, vehicle group.

FIG. 23 in two panels contains photomicrographs that illustrate thatCompound C0105 treatment of mice dramatically reduces NFTimmunostaining. Representative sections immunostained with ananti-phospho-tau antibody (FIG. 23A) clearly show that Compound C0105treatment greatly reduced NFT immunoreactivity in both prefrontal cortex(FCX) and hippocampus (HP) of mice receiving ICV Aβ₄₂ infusions.Quantitation of optical density in all animals (FIG. 23B) shows thatCompound C0105 significantly reduced Aβ₄₂-induced NFT immunoreactivityin both regions. #p<0.01 Aβ₄₂ vs. Aβ₄₂+C0105, *p<0.01 vs. sham.

FIG. 24 in two panels contains photomicrographs that illustrate thatCompound C0105 treatment of mice dramatically reduces immunostaining ofAβ₄₂ aggregates. Representative sections immunostained for Aβ₄₂aggregates (FIG. 24A) clearly show that Compound C0105 treatment greatlyreduced immunostaining of Aβ₄₂ aggregates in both prefrontal cortex(FCX) and hippocampus (HP) of mice receiving ICV Aβ₄₂ infusions.Quantitation of optical density in all animals (FIG. 24B) shows thatCompound C0105 significantly reduced Aβ₄₂-induced NFT immunoreactivityin both regions. #p<0.01 Aβ₄₂ vs. Aβ₂+C0105, *p<0.01 vs. sham.

FIG. 25 in five panels illustrates that Compound C0105 decreasedAβ₄₂-induced FLNA association with both α7nAChR and TLR4 in humanpostmortem AD and control brain tissue. AD and age-matched control brainslices were treated with 0.1 or 1 nM concentrations of Compound C0105,and control brain slices were simultaneously treated with Aβ₄₂. Theextent of FLNA association with α7nAChR or TLR4 was assessed in thesolubilized synaptosomes by immunoprecipitating with immobilizedanti-FLNA and Western blot detection (FIG. 25A) using antibodiesspecific to each receptor, and wherein numerals outside of and to theleft of the blots are as discussed before. Blots were analyzed bydensitometric quantitation (FIG. 25B and FIG. 25D). AD tissue andAβ₄₂-treated control tissue showed a markedly increased association ofα7nAChR and TLR4 with FLNA, and Compound C0105 reduced theseassociations. Percent inhibition is depicted in FIG. 25C and FIG. 25E.n=11. Data are means±SEM. *p<0.01 vs. vehicle-treated control, #p<0.01vs. Aβ₄₂-treated control or vehicle-treated AD.

FIG. 26 in two panels illustrates that Compound C0105 reducesAβ₄₂-α7nAChR complexes in AD and Aβ₄₂-treated control. Solubilizedsynaptosomes from the same treated AD and control brain slices used inFIG. 25 were immunoprecipitated with anti-Aβ₄₂ and Western blots (FIG.26A) of immunoprecipitates were probed with anti-α7nAChR and analyzed bydensitometric quantitation (FIG. 26B). Aβ₄₂-α7nAChR complexes wereelevated in both Aβ₄₂-treated control tissue and AD tissue, and CompoundC0105 (0.1 and 1 nM) reduced this interaction. n=11. Data are means±SEM.*p<0.01 vs. vehicle-treated control, #p<0.01 vs. Aβ₄₂-treated control orvehicle-treated AD.

FIG. 27 in two panels illustrates that Compound C0105 reduces affinityof the Aβ₄₂-α7nAChR interaction. In postmortem control tissue, CompoundC0105 incubation reduces Aβ₄₂ binding affinity for α7nAChR 1000-foldfrom 100 femtomolar to 16 nanomolar in biotinylated synaptic membranesfrom postmortem frontal cortices of non-demented controls (FIG. 27A). Infresh, SK-N-MC cells, Compound C0105 reduces this binding affinity10,000-fold from 770 femtomolar to 1 nanomolar (FIG. 27B). Data aremeans±SEM. n=11 for postmortem control tissue; n=6 for SK-N-MC cells.

FIG. 28 is a bar graph that illustrates that Compound C0105 reducesα7nAChR dysfunction. AD brain slices and Aβ₄₂-treated control brainslices had significantly impaired calcium influx after stimulating withthe α7nAChR full agonist PNU282987. Compound C0105 treatment normalizedthis impairment. Data are means±SEM. n=11. *p<0.01 vs. vehicle-treatedcontrol, #p<0.01 vs. vehicle-treated AD group, +p<0.01 vs. vehicle- andCompound C0105-treated control groups.

FIG. 29 is a bar graph that illustrates that Compound C0105 reducesNMDAR dysfunction. AD brain slices and Aβ₄₂-treated control brain sliceshad significantly impaired Ca⁺² influx after stimulating with NMDA andglycine, co-agonists of NMDAR. Compound C0105 treatment normalized thisimpairment. Data are means±SEM. n=11. *p<0.01 vs. basal level incontrol, #p<0.01 vs. vehicle-treated AD group, +p<0.01 compared tovehicle- and Compound C0105-treated control groups.

FIG. 30 is a bar graph that illustrates that 1 nM Compound C0105partially normalizes K⁺-evoked Ca⁺² influx. K⁺-evoked Ca⁺² influx isdramatically decreased in AD brain slices and in Aβ₄₂-treated controlbrain slices, indicating nonfunctioning cells or cell death. CompoundC0105 incubation significantly elevates this depolarization-induced Ca⁺²influx, rescuing some of the nonfunctioning cells. Data are means±SEM.n=11. *p<0.01 vs. vehicle-treated control group; #p<0.01 vs.vehicle-treated AD group; +p<0.01 vs. vehicle- and CompoundC0105-treated control groups.

FIG. 31 in two panels illustrates that Compound C0105 normalizes NMDARsignaling impairments. NMDAR dysfunction in AD or Aβ₄₂-treated controlbrain slices is also evidenced by reductions in linkages of severalsignaling molecules to NR-1, the obligatory NMDAR subunit. CompoundC0105 (1 nM) mitigates these reductions. Western blots (FIG. 31A), inwhich numerals outside of and to the left of the blots are as discussedbefore, were analyzed by densitometric quantitation (FIG. 31B). Data aremeans±SEM. n=11. *p<0.01 vs. NMDA/glycine-stimulated level invehicle-treated control group; #p<0.01 vs. NMDA/glycine-stimulated levelin vehicle-treated AD group.

FIG. 32 in two panels illustrates that Compound C0105 normalizes IRsignaling impairments. IR signaling is impaired in AD and Aβ₄₂-treatedcontrol brain slices, as measured by phosphorylation of IPβ and itsassociation with the signaling molecule IRS-1. Incubation with CompoundC0105 (1 nM) normalizes these impairments. Western blots (FIG. 32A), inwhich numerals outside of and to the left of the blots are as discussedbefore, were analyzed by densitometric quantitation (FIG. 32B). Data aremeans±SEM. n=11. *p<0.01 compared to insulin-stimulated level in controlvehicle group; #p<0.01 vs. insulin-stimulated level in AD vehicle group.

FIG. 33 in two panels illustrates that VAKGL (SEQ ID NO: 1) pentapeptide(10 μM) blocks C0105's prevention of FLNA-α7nAChR or TLR4 association.Acting as a decoy for the FLNA protein, the pentapeptide binding site ofCompound C0105 on FLNA blocks Compound C0105's reduction in Aβ₄₂-inducedFLNA-α7nAChR/TLR4 association in postmortem frontal corticalsynaptosomes. Western blots (FIG. 33A), in which numerals outside of andto the left of the blots are as discussed before, were analyzed bydensitometric quantitation (FIG. 33B). Data are means±SEM. n=3. *p<0.01vs. the respective basal level; #p<0.01 vs. Aβ₄₂-exposed tissues.

FIG. 34 VAKGL (SEQ ID NO: 1) pentapeptide (10 μM) blocks CompoundC0105's prevention of tau phosphorylation. Again using postmortemfrontal cortical synaptosomes, the VAKGL pentapeptide blocks C0105'sreduction in tau phosphorylation at all three phosphorylation sitesfound in neurofibrillary tangles. Western blots (FIG. 34A), in whichnumerals outside of and to the left of the blots are as discussedbefore, were analyzed by densitometric quantitation (FIG. 34B). Data aremeans±SEM. n=3. *p<0.01 vs. the respective basal level; #p<0.01 vs.Aβ₄₂-exposed tissues.

FIG. 35 through FIG. 40 represent schematic pharmacophores(Pharmacophores 1-6, respectively) showing relative locations ofchemical features such as a hydrogen bond acceptor (HBA), anaromatic/hydrophobe (ARO/HYD) center, and the intramolecular distancesthere between in Ångstroms for a compound that binds to the pentamericpeptide of FLNA of SEQ ID NO: 1.

FIG. 41, in two panels, FIGS. 41 and 41B, illustrates furtherhigh-affinity FLNA-binding compounds that reduce α7nAChR-FLNAassociation assayed as in FIG. 2. Frontal cortical synaptosomes from2-month-old rats were treated with 1 or 10 nM concentrations ofCompounds A, B, or C, using Compounds C0134 and C0105 as controls,either simultaneously (Sim) with or 10 minutes prior (10′ pr) to Aβ₄₂(0.1 μM) and were immunoprecipitated with immobilized anti-FLNA. Thecomplexes in the solubilized synaptosomes, and α7nAChR, TLR4 and FLNAlevels in the anti-FLNA immunoprecipitates were determined by Westernblotting (FIG. 41A) in which numerals outside of and to the left of theblots are as discussed before. Amounts present in the blots werequantified by densitometry (FIG. 41B). Ratios of α7nAChR/FLNA werestatistically different from Aβ₄₂ alone with p<0.01 for all compoundsexamined Dunnett's test. Ratios of TLR4/FLNA were statisticallydifferent from Aβ₄₂ alone for most of the compounds and conditions at*p<0.01 or **p<0.05. Structural formulas of the compounds used in thisand the other figures are provided hereinafter.

FIG. 42, also in two panels, FIGS. 42A and 42B, illustrates thatFLNA-binding compounds reduce tau phosphorylation at all threephosphorylation sites using the compounds and concentrations of FIG. 41using immunoprecipitation and Western blotting. Thus, in the sametreated synaptosomes used in FIG. 41, levels of tau proteinphosphorylated at S²⁰², T²³¹ and T¹⁸¹ were measured inimmunoprecipitates using an anti-tau antibody that does not distinguishits phosphorylation state (Tau). The three phosphoepitopes of tau weredetected in immunoprecipitates using specific antibodies. Aβ₄₂ stronglypromotes tau phosphorylation at all three sites (FIG. 42A).Densitometric analysis of the blots (FIG. 42B) showed that all fivecompounds reduced this phosphorylation at 10 nM concentration eitherwhen simultaneously administered, or with 10-minute pretreatment at bothconcentrations compared to Aβ₄₂ alone using Dunnett's test at *p<0.01.Compound A also reduced tau phosphorylation with simultaneousadministration at 1 nM with **p<0.05 or *p<0.01, whereas Compound C didnot provide a statistically significant result at two phosphorylationpositions when simultaneously administered.

FIG. 43, in two panels, FIG. 43A and FIG. 43B, illustrates furtherhigh-affinity FLNA-binding compounds that reduce α7nAChR-FLNAassociation assayed as in FIG. 2. Frontal cortical synaptosomes from2-month-old rats were treated with 1 or 10 nM concentrations of CompoundC0087 and Compound C0108, using Compound C0105 as a control, eithersimultaneously (Sim) or 10 minutes prior (10′ pr) to Aβ₄₂ (0.1 μM) andwere immunoprecipitated with immobilized anti-FLNA. The complexes in thesolubilized synaptosomes, and α7nAChR, TLR4 and FLNA levels in theanti-FLNA immunoprecipitates were determined by Western blotting (FIG.43A) in which numerals outside of and to the left of the blots are asdiscussed before. Amounts present in the blots were quantified bydensitometry (FIG. 43B). Ratios of α7nAChR/FLNA and TLR4/FLNA werestatistically different from Aβ₄₂ alone with **p<0.05, *p<0.01 for thecompounds as shown using Dunnett's test. Structural formulas of thecompounds used in this and the other figures are provided hereinafter.

FIG. 44, also in two panels, FIG. 44A and FIG. 44B, illustrates thatFLNA-binding compounds reduce tau phosphorylation at all threephosphorylation sites using the compounds and concentrations of FIG. 43using immunoprecipitation and Western blotting. Thus, in the sametreated synaptosomes used in FIG. 43, levels of tau proteinphosphorylated at S²⁰², T²³¹ and T¹⁸¹ were measured inimmunoprecipitates using an anti-tau antibody that does not distinguishits phosphorylation state (Tau). The three phosphoepitopes of tau weredetected in immunoprecipitates using specific antibodies. Aβ₄₂ stronglypromotes tau phosphorylation at all three sites. Densitometric analysisof the blots (FIG. 44B) showed that both compounds reduced thisphosphorylation at both concentrations either simultaneouslyadministered or with 10-minute pretreatment compared to Aβ₄₂ alone usingDunnett's test at **p<0.05, *p<0.01.

FIG. 45, in two panels, FIG. 45A and FIG. 45B, illustrates furtherhigh-affinity FLNA-binding compounds that reduce α7nAChR-FLNAassociation assayed as in FIG. 2. Frontal cortical synaptosomes from2-month-old rats were treated with 1 or 10 nM concentrations of CompoundC0124, using Compound C0105 as a control, either simultaneously (Sim) or10 minutes prior (10′ pr) to Aβ₄₂ (0.1 μM) and were immunoprecipitatedwith immobilized anti-FLNA. The complexes in the solubilizedsynaptosomes, and α7nAChR, TLR4 and FLNA levels in the anti-FLNAimmunoprecipitates were determined by Western blotting (FIG. 45A) inwhich numerals outside of and to the left of the blots are as discussedbefore. Amounts present in the blots were quantified by densitometry(FIG. 45B). Ratios of α7nAChR/FLNA and TLR4/FLNA were statisticallydifferent from Aβ₄₂ alone with **p<0.05, *p<0.01 for the compounds asshown using Dunnett's test. Structural formulas of the compounds used inthis and the other figures are provided hereinafter.

FIG. 46, also in two panels, FIG. 46A and FIG. 46B, illustrates thatFLNA-binding compounds reduce tau phosphorylation at all threephosphorylation sites using the compounds and concentrations of FIG. 45using immunoprecipitation and Western blotting. Thus, in the sametreated synaptosomes used in FIG. 45, levels of tau proteinphosphorylated at S²⁰², T²³¹ and T¹⁸¹ were measured inimmunoprecipitates using an anti-tau antibody that does not distinguishits phosphorylation state (Tau). The three phosphoepitopes of tau weredetected in immunoprecipitates using specific antibodies. Aβ₄₂ stronglypromotes tau phosphorylation at all three sites. Densitometric analysisof the blots (FIG. 46B) showed that both of Compounds C0105 and C0124reduced this phosphorylation at both concentrations eithersimultaneously administered or with 10-minute pretreatment compared toAβ₄₂ alone using Dunnett's test at *p<0.01.

FIG. 47, in three panels as FIGS. 47A-47C, illustrates that LPS at eachof two concentrations (10 μg/ml and 10 μg/ml) induces tauphosphorylation at all three phosphorylation sites in human postmortemhippocampal cell slices, and that that phosphorylation is inhibited bycompound C0105 at a concentration of 1 nM using immunoprecipitation andWestern blotting. The levels of tau protein phosphorylated at S²⁰², T²³¹and T¹⁸¹ were measured in immunoprecipitates using an anti-tau antibodythat does not distinguish its phosphorylation state (Tau). The threephosphoepitopes of tau were detected in immunoprecipitates usingspecific antibodies using Western blots (WB). Aβ₄₂ was used as a controlthat also promotes tau phosphorylation at all three sites. Densitometricanalysis of the blots (FIGS. 47B and 47C) provides a quantitativeillustration that Compound C0105 reduced this phosphorylation usingNewman-Keuls multiple comparisons: *p<0.01, **p<0.05 compared tovehicle-incubated group, and #p<0.01 compared to respective LPS or Ab₄₂treated group. The graphs in FIG. 47C are in the same order as thoseshown in FIG. 47B.

FIG. 48, in four parts as FIG. 48A, 48B, 48C and 48D, are graphs showingcompetition curves that illustrate the binding of radio-labeled naloxone[³H]NLX in the presence of naltrexone (NTX) or illustrative CompoundC0105 to the filamin A (FLNA) or the filamin A (FLNA) pentamer of SEQ IDNO. 1 as reported in Wang et al., PLoS One. 3 (2):e1554 (2008). FIG. 48Aillustrates [³H]NLX binding to FLNA in the membranes of A7 cells in thepresence of indicated amounts of naltrexone (NTX) and is taken from Wanget al., PLoS One. 3 (2):e1554 (2008), FIG. 3; FIG. 48B illustratesbinding of [³H]NLX to FLNA in the membranes of A7 cells in the presenceof indicated amounts of Compound C0105; FIG. 48C. illustrates binding of[³H]NLX in the presence of indicated amounts of Compound C0105 to FLNAin the membranes of SK-N-MC cells; and FIG. 48D illustrates binding of[³H]NLX to the FLNA pentamer of SEQ ID NO. 1 in the presence ofindicated amounts of Compound C0105.

Abbreviations and Short Forms

The following abbreviations and short forms are used in thisspecification.

“Aβ” means amyloid-beta

“Aβ₄₂” means a 42-residue proteolysis product of amyloid precursorprotein (APP)

“α7nAchR” means alpha-7 nicotinic acetylcholine receptor

“DAMGO” means [D-Ala2, N-MePhe4, Gly-ol]-enkephalin

“ERK2” means extracellular signal-regulated kinase 2

“FCX” means frontal cortex or prefrontal cortex

“FLNA” means filamin A

“FITC” means fluorescein isothiocyanate

“Gs” means G protein stimulatory subtype, stimulates adenylyl cyclase

“HP” means hippocampus

“IHC” means immunohistochemistry

“IR” means insulin receptor

“MOR” means μ opioid receptor

“NLX” means naloxone

“NTX” means naltrexone

“NFTs” means neurofibrillary tangles

“NMDA” means N-methyl-D-aspartate

“NMDAR” means NMDA receptor

“pERK2” means phosphorylated ERK2

“pTau” means hyperphosphorylated tau protein

“TLR4” means toll-like receptor-4

DEFINITIONS

In the context of the present invention and the associated claims, thefollowing terms have the following meanings:

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “hydrocarbyl” is a short hand term for anon-aromatic group that includes straight and branched chain aliphaticas well as alicyclic groups or radicals that contain only carbon andhydrogen. Inasmuch as alicyclic groups are cyclic aliphatic groups, suchsubstituents are deemed hereinafter to be subsumed within the aliphaticgroups. Thus, alkyl, alkenyl and alkynyl groups are contemplated,whereas aromatic hydrocarbons such as phenyl and naphthyl groups, whichstrictly speaking are also hydrocarbyl groups, are referred to herein asaryl groups, substituents, moieties or radicals, as discussedhereinafter. An aralkyl substituent group such as benzyl is deemed anaromatic group as being an aromatic ring bonded to an X group, where Xis CH₂. A substituent group containing both an aliphatic ring and anaromatic ring portion such as tetralin (tetrahydronaphthalene) that islinked directly through the aliphatic portion to the depicted ringcontaining the W group is deemed a non-aromatic, hydrocarbyl group. Onthe other hand, a similar group bonded directly via the aromaticportion, is deemed to be a substituted aromatic group. Where a specificaliphatic hydrocarbyl substituent group is intended, that group isrecited; i.e., C₁-C₄ alkyl, methyl or dodecenyl. Exemplary hydrocarbylgroups contain a chain of 1 to about 12 carbon atoms, and preferably 1to about 8 carbon atoms, and more preferably 1 to 6 carbon atoms.

A particularly preferred hydrocarbyl group is an alkyl group. As aconsequence, a generalized, but more preferred substituent can berecited by replacing the descriptor “hydrocarbyl” with “alkyl” in any ofthe substituent groups enumerated herein.

Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl,octyl, decyl, dodecyl and the like. Cyclic alkyl radicals such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl arealso contemplated, as are their corresponding alkenyl and alkynylradicals. Examples of suitable straight and branched chain alkenylradicals include ethenyl (vinyl), 2-propenyl, 3-propenyl,1,4-pentadienyl, 1,4-butadienyl, 1-butenyl, 2-butenyl, 3-butenyl,decenyl and the like. Examples of straight and branched chain alkynylradicals include ethynyl, 2-propynyl, 3-propynyl, decynyl, 1-butynyl,2-butynyl, 3-butynyl, and the like.

Usual chemical suffix nomenclature is followed when using the word“hydrocarbyl” except that the usual practice of removing the terminal“yl” and adding an appropriate suffix is not always followed because ofthe possible similarity of a resulting name to one or more substituents.Thus, a hydrocarbyl ether is referred to as a “hydrocarbyloxy” grouprather than a “hydrocarboxy” group as may possibly be more proper whenfollowing the usual rules of chemical nomenclature. Illustrativehydrocarbyloxy groups include methoxy, ethoxy, and cyclohexenyloxygroups. On the other hand, a hydrocarbyl group containing a—C(O)-functionality is referred to as a hydrocarboyl (acyl) and thatcontaining a —C(O)O— is a hydrocarboyloxy group inasmuch as there is noambiguity. Exemplary hydrocarboyl and hydrocarboyloxy groups includeacyl and acyloxy groups, respectively, such as acetyl and acetoxy,acryloyl and acryloyloxy.

Carboxyl-related linking groups between the central spiro ring systemand an aromatic or heteroaromatic ring system, circle A, include severaltypes of ester and amide bonds. Illustrative of such bonds aresulfonamide, sulfonate and thiosulfonate esters that can be formedbetween a SO₂-containing group [also sometimes shown as a S(═O)₂ group]and an amine, oxygen or sulfur atom, respectively. Amide, ester andthioester links can be formed between an aromatic or heteroaromatic ringcontaining a C(O) [also sometimes shown as (C═O)] group and a nitrogen,oxygen or sulfur atom, respectively. Similarly, a guanidino linker canbe formed between an aromatic or heteroaromatic ring containing aNHC(NH) [NHC(═NH)] group and a nitrogen, a urethane, carbonate orthiocarbonate can be formed between an aromatic or heteroaromatic ringcontaining a OC(O) [or OC(═O)] group and a nitrogen, oxygen or sulfur,respectively. A compound containing a urea linker, urethane linker orisothiourea linker [NHC(O)S] {or [NHC(═O)S]} can be formed between anaromatic or heteroaromatic ring containing a NHC(O) group and anitrogen, oxygen or sulfur, respectively. A thiourea linkage is alsocontemplated.

A “carboxyl” substituent is a —C(O)OH group. A C₁-C₆ hydrocarbylcarboxylate is a C₁-C₆ hydrocarbyl ester of a carboxyl group. Acarboxamide is a —C(O)NR³R⁴ substituent, where the R groups are definedelsewhere and are numbered here as 3 and 4 for ease in furtherdiscussion, but need not be so numbered in the following chemicalformulas. Similarly, a sulfonamide is a —S(O)₂NR³R⁴ substituent, wherethe R groups are defined hereinafter. Illustrative R³ and R⁴ groups thattogether with the depicted nitrogen of a carboxamide form a 5-7-memberedring that optionally contains 1 or 2 additional hetero atoms thatindependently are nitrogen, oxygen or sulfur, include morpholinyl,piperazinyl, oxathiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyrazolyl,1,2,4-oxadiazinyl and azepinyl groups.

As a skilled worker will understand, a substituent that cannot existsuch as a C₁ alkenyl or alkynyl group is not intended to be encompassedby the word “hydrocarbyl”, although such substituents with two or morecarbon atoms are intended.

The term “aryl”, alone or in combination, means a phenyl, naphthyl orother radical as recited hereinafter that optionally carries one or moresubstituents selected from hydrocarbyl, hydrocarbyloxy, halogen,hydroxy, amino, nitro and the like, such as phenyl, p-tolyl,4-methoxyphenyl, 4-(tert-butoxy)phenyl, 4-fluorophenyl, 4-chlorophenyl,4-hydroxyphenyl, and the like. The term “arylhydrocarbyl”, alone or incombination, means a hydrocarbyl radical as defined above in which onehydrogen atom is replaced by an aryl radical as defined above, such asbenzyl, 2-phenylethyl and the like. The term“arylhydrocarbyloxycarbonyl”, alone or in combination, means a radicalof the formula —C(O)—O-arylhydrocarbyl in which the term“arylhydrocarbyl” has the significance given above. An example of anarylhydrocarbyloxycarbonyl radical is benzyloxycarbonyl. The term“aryloxy” means a radical of the formula aryl-O— in which the term arylhas the significance given above. The term “aromatic ring” incombinations such as substituted-aromatic ring sulfonamide,substituted-aromatic ring sulfinamide or substituted-aromatic ringsulfenamide means aryl or heteroaryl as defined above.

As used herein, the term “binds” refers to the specific adherence ofmolecules to one another, such as, but not limited to, the interactionof a ligand with its receptor, or a polypeptide of SEQ ID NO: 1 with asmall molecule such as the compounds disclosed herein, or an antibodyand its antigen.

As used herein, the term “FLNA-binding compound” refers to a compoundthat binds to the scaffolding protein filamin A, or more preferably to apolypeptide comprising residues -Val-Ala-Lys-Gly-Leu- (SEQ ID NO: 1) ofthe FLNA sequence that correspond to amino acid residue positions2561-2565 of the FLNA protein sequence as noted in the sequence providedat the web address: UniProtKB/Swiss-Prot entry P21333, FLNA-HUMAN,Filamin-A protein sequence. A FLNA-binding compound can inhibit theMOR-Gs coupling caused by agonist stimulation of the p opioid receptorvia interactions with filamin A, preferably in the 24^(th) repeatregion.

As used herein, the term “opioid receptor” refers to a G protein-coupledreceptor located in the CNS that interacts with opioids. Morespecifically, the p opioid receptor is activated by morphine causinganalgesia, sedation, nausea, and many other side effects known to one ofordinary skill in the art.

As used herein, the term “opioid agonist” refers to a substance thatupon binding to an opioid receptor can stimulate the receptor, induce Gprotein coupling and trigger a physiological response. Morespecifically, an opioid agonist is a morphine-like substance thatinteracts with MOR to produce analgesia.

As used herein, the term “opioid antagonist” refers to a substance thatupon binding to an opioid receptor inhibits the function of an opioidagonist by interfering with the binding of the opioid agonist to thereceptor.

As used herein the term “ultra-low-dose” or “ultra-low amount” refers toan amount of compound that when given in combination with an opioidagonist is sufficient to enhance the analgesic potency of the opioidagonist. More specifically, the ultra-low-dose of an opioid antagonistis admixed with an opioid agonist in an amount about 1000- to about10,000,000-fold less, and preferably about 10,000- to about1,000,000-fold less than the amount of opioid agonist.

As used herein an “FLNA-binding effective amount” or more simply an“effective amount” refers to an amount of a contemplated compoundsufficient to bind to the FLNA pentapeptide of SEQ ID NO: 1 and performthe functions described herein, such as inhibition of tau proteinphosphorylation. An effective amount of a contemplated compound is mosteasily determined using the in vitro assay of Example 1. Using thatdefinition, an effective amount of a contemplated compound binds to apentapeptide of SEQ ID NO: 1, inhibits at least about 60 percent andmore preferably about 70 percent of the FITC-labeled naloxone bindingwhen present at a 10 μM concentration and using unlabeled naloxone asthe control inhibitor at the same concentration and under the sameconditions as the contemplated compound, and up to about twice (200percent) the inhibition obtained with naloxone as control.

As used herein the term “pharmacophore” is not meant to imply anypharmacological activity. A pharmacophore can be defined as the relevantgroups on a molecule that interact with a receptor and are responsiblefor the activity of the compound. [R. B. Silverman, The OrganicChemistry of Drug Design and Drug Action, 2^(nd) ed., Elsevier AcademicPress, Amsterdam, (2004), p. 17.] The term can also be defined and isintended herein to be the chemical features of a molecule and theirdistribution in three-dimensional space that constitutes the preferredrequirements for molecular interaction with a receptor (See, U.S. Pat.No. 6,034,066). A pharmacophore is computer-calculated by determiningthe shared aromatic/hydrophobic and hydrogen bond acceptor functions andthe distances there between of a group of compounds that bind similarlyto a particular receptor, here, pentapeptide of SEQ ID NO: 1, using anappropriately programmed computer. Such computer programs are availablecommercially from companies such as Accelrys Software Inc., San Diego,Calif., Schrödinger, LLC, Portland, Oreg., from Chemical ComputingGroup, Inc., Montreal, QC, Canada, or as an open access program referredto as ZINCPharmer

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates a method of inhibiting thehyperphosphorylation (phosphorylation) of tau protein at one or more ofS²⁰², T²³¹ and T¹⁸¹ in vitro as well as, in vivo. Such a methodcomprises the steps of administering to cells of the central nervoussystem in recognized need thereof, such as brain cells, an effectiveamount of a compound that binds to the FLNA pentapeptide of SEQ ID NO:1, inhibits at least about 60 percent and more preferably about 70percent of the FITC-labeled naloxone binding when present at a 10 μMconcentration and using unlabeled naloxone as the control inhibitor atthe same concentration. That compound also preferably contains at leastfour of the six pharmacophores of FIGS. 35-40. A more preferred compoundcontains five of the six pharmacophores, and a most preferred compoundcontains all six of those pharmacophores.

Phosphorylation of one or more of S²⁰², T²³¹ and T¹⁸¹ of the tau proteinis typically in addition to phosphorylation of one or more additionalresidues of the protein. The presence of such phosphorylation can bedetermined by the immunoreaction of an antibody, usually a monoclonalantibody, that immunoreacts specifically with a tau protein that isphosphorylated at one of those three amino acid residues as isillustrated herein.

The administration is preferably carried out in the absence of aMOR-binding effective amount of a separate, exogenously provided MORagonist or antagonist molecule. Thus, an exogenously suppliedMOR-binding compound such as morphine itself, codeine, oxycodone and thelike MOR-binding compounds is absent when a contemplated compound isadministered to the cells. The presence of an endogenously suppliedMOR-binding compound such as an endorphin or an enkephalin cannot be asreadily controlled and is not excluded. Some of the contemplated FLNApentapeptide-binding compounds also bind to MOR and their use is alsonot excluded. However, it is preferred to use a compound that bindspoorly if at all to MOR, as discussed hereinafter, and is not a MORagonist. Such a compound exhibits less than about 80 percent the MORstimulation provided by DAMGO at the same concentration and assayconditions.

Inhibition of the hyperphosphorylation (phosphorylation) of tau proteinin vitro and in vivo can be assayed in lysates or other cellpreparations of cultured CNS cells in vitro or in lysates of CNS cellssuch as olfactory neurons obtained via scraping the nasal cavity forneural epithelial cells or from biopsy samples for in vivo assays.

Also contemplated is a method of inhibiting TLR4-mediated immuneresponse such as inflammation of TLR4-containing cells such aslymphocytes or cells of the CNS that comprises administering toTLR4-containing cells in recognized need thereof an effective amount ofa of a compound or a pharmaceutically acceptable salt thereof that bindsto a pentapeptide of filamin A (FLNA) of SEQ ID NO: 1, inhibits at leastabout 60 percent and more preferably about 70 percent of theFITC-labeled naloxone binding when present at a 10 μM concentration andusing unlabeled naloxone as the control inhibitor at the sameconcentration, and contains at least four of the six pharmacophores ofFIGS. 35-40. The administration is preferably carried out in the absenceof a mu opioid receptor (MOR)-binding effective amount of a separate MORagonist or antagonist molecule. An administered compound preferablybinds poorly or does not bind to MOR as described hereinafter, e.g., thecompound exhibits less than about 80 percent the MOR stimulationprovided by DAMGO at the same concentration.

The use of a single stereoisomer or mixture of stereoisomers, or apharmaceutically acceptable salt of a contemplated compound is alsocontemplated. The contemplated administration can take place in vivo orin vitro, and is typically repeated a plurality of times over a periodof days or months when administered in vivo to the cells of a hostanimal such as a human.

An administered compound contains at least four of the sixpharmacophores of FIGS. 35-40, but more preferably, such a contemplatedcompound contains at least five of the six pharmacophores of FIGS.35-40. Most preferably, the administered compound contains each of thesix pharmacophores of FIGS. 35-40.

TLR4-mediated immune response inflammation of CNS cells produceshyperphosphorylation of the tau protein and related tauopathies such asthose that result from NFTs. As a consequence, one way to assay for thepresence of TLR4-mediated inflammation is to assay for the presence ofan enhanced amount of phosphorylated tau compared to the amount presentin a non-inflammatory condition as was described above forhyperphosphorylated tau.

TLR4-mediated inflammation can also be recognized by the greater thanbackground abundance of TLR4 activation protein markers such as thecytokines IL-1β, IL-6 and TNFα that are typically enhanced together,and/or the separately stimulated NF-κB and JNK proteins. As was notedearlier, enhanced expression of IL-1β, IL-6 and TNFα as compared toexpression of NF-κB and JNK appear to proceed by different TLR4-mediatedpathways. However, both markers of inflammation can be present at thesame time due to the same immunostimulus.

Thus, the presence of an enhanced amount of one, two or three of IL-1β,IL-6 and TNFα relative to the amount present in a non-inflammatorycondition indicates the presence of TLR4-mediated inflammation.Similarly, the enhanced presence of the transcription factor NF-κB andthe mitogen-activated protein kinase c-Jun N-terminal kinase (JNK)compared to the amount present in a non-inflammatory conditionseparately implies the presence of TLR4-mediated inflammation.

These proteins or polypeptides can be assayed in lysates of culturedcells such as lymphocytes such as B cells, T cells and macrophages orCNS cells such as olfactory neurons that can be obtained by scraping thenasal cavity for neural epithelial cells for in vivo assays. Theproteins can also be assayed in the cell culture medium for in vitrostudies using lymphocytes or CNS cells such as those illustratedhereinafter and in body fluids such as blood or its constituent plasmaor serum or lymphocytes for in vivo assays.

Administration of a contemplated compound or its pharmaceuticallyacceptable salt is continued until tau hyperphosphorylation is no longerenhanced and/or until the amount of one or more of the TLR4 activationprotein markers is at background levels. Enhancement of the level ofhyperphosphorylated tau or one of the TLR4 protein markers relative tobackground (in the absence of a TLR4-mediated immune response) conditionis determined by a difference that is statistically significant at leastat the 90 percent confidence level (p<0.1), and preferably at the 95percent confidence level (p<0.05) as are illustrated in the accompanyingfigures.

It is also preferred that an administered compound or a pharmaceuticallyacceptable salt thereof be present dissolved or dispersed in apharmaceutically acceptable diluent as a pharmaceutical composition whenadministered. Most preferably, the administration is peroral.

The use of a pharmaceutically acceptable salt of a contemplated compoundis also contemplated, as is the use of a single stereoisomer or mixtureof stereoisomers, or of their pharmaceutically acceptable salts. Thecontemplated administration can take place in vivo or in vitro.

In presently preferred embodiments, the present invention contemplates amethod of inhibiting 1) phosphorylation of the tau protein and/or 2)TLR4-mediated immune response (e.g., inflammation) of lymphocytes and/orcells of the CNS that comprises administering to cells of the centralnervous system in recognized need thereof such as brain cells aneffective amount of a compound of one or more of Series A, Series B,Series C-1, Series C-2, Series D and Series E, single enantiomer, amixture of enantiomers or a pharmaceutically acceptable salt of anycontemplated compound(s). The administration is preferably carried outin the absence of a MOR-binding effective amount of a separate MORagonist or antagonist molecule. Illustrative of CNS cells are cells suchas those of a host animal that exhibit inflammation induced by braininjury such as traumatic brain injury, chronic traumatic encephalopathy,as well as those of a host animal such as a human exhibiting Alzheimer'sdisease (AD) symptoms, frontotemporal dementia (FTD), progressivesupranuclear palsy, dementia pugilistica and corticobasal degeneration,as well as infection by Gram positive and/or Gram negative bacteria.

The general structures of the compounds of each series are shown below.A detailed discussion of compounds of each of those series is set out ina section entitled “Contemplated Compounds” that follows.

In accordance with a method described above, a composition that containsan effective amount of a contemplated compound or its pharmaceuticallyacceptable salt dissolved or dispersed in a pharmaceutically acceptablediluent is administered to cells of the CNS in recognized need thereof,and particularly the brain, in vivo in a living animal or in vitro in acell preparation. When administered in vivo to an animal such as alaboratory rat or mouse or a human in recognized need, theadministration inhibits the formation of phosphorylated-tau-containingNFTs in the CNS such as in the brain of a subject animal to which acontemplated compound is administered. Admixture of a compositioncontaining a contemplated compound or its pharmaceutically acceptablesalt dissolved or dispersed in a pharmaceutically acceptable diluentwith CNS cells such as a brain cell preparation in vitro also inhibitsthe formation of NFTs as is illustrated hereinafter.

A contemplated compound binds to the scaffolding FLNA protein, andparticularly to a five residue portion of the FLNA protein sequenceVal-Ala-Lys-Gly-Leu (SEQ ID NO: 1) in an in vitro assay that isdiscussed hereinafter in Example 1, and briefly below. A contemplatedcompound binds only to a single site on FLNA and that site is the SEQ IDNO: 1 pentapeptide site.

Binding studies of the naltrexone inhibition of tritiated-naloxone,[³H]NLX, binding to membranes from FLNA-expressing A7 cells (anastrocyte cell line produced by immortalizing optic nerve astrocytesfrom the embryonic Sprague-Dawley rat with SV40 large T antigen) hasshown the existence of two affinity sites on FLNA; a high affinity site(H) with an IC₅₀—H of 3.94 picomolar and a lower affinity site (L)IC₅₀-L of 834 picomolar. [Wang et al., PLoS One. 3 (2):e1554 (2008);Wang et al., PLoS One. 4 (1):e4282 (2009).] The high affinity site wassubsequently identified as the FLNA pentapeptide of SEQ ID NO: 1 (USPatent Publication 2009/0191579 and its predecessor application Ser. No.60/985,086 that was filed on Nov. 2, 2007), whereas the lower affinitysite has not yet been identified.

Compounds such as naloxone (NLX), naltrexone (NTX), methadone, fentanyl,nalorphine, nalbuphine and buprenorphine, and the like bind well to thehigh affinity FLNA pentapeptide of SEQ ID NO: 1 (VAKGL). However, whenused at a dosage recited on the product label, those compounds also bindto the lower affinity site on FLNA, and typically also bind to the MOR.Some of the compounds are MOR antagonists such as naloxone, naltrexone,nalbuphine, whereas others such as methadone, buprenorphine and fentanylare full or partial agonists of MOR. Binding to that lower affinity FLNAsite impairs the activity of the FLNA pentapeptide of SEQ ID NO: 1 toexhibit its activities as discussed, utilized and illustrated herein. Asa consequence, compounds such as naloxone, naltrexone, methadone,fentanyl, nalorphine, nalbuphine, buprenorphine and similar compoundsthat also bind to the lower affinity site on the FLNA protein are notcontemplated for use herein.

A compound contemplated for use in the present invention inhibits thebinding of fluorescein isothiocyanate-labeled naloxone (FITC-NLX) tobiotin-linked SEQ ID NO: 1 (Bn-VAKGL) bound to coated streptavidinplates under conditions defined hereinafter in Example 1 to an extentthat is at least about 60 percent and more preferably at least about 80percent of the value obtained of the value obtained when present at a 10μM concentration and using naloxone as the control inhibitor at the sameconcentration as the contemplated compound, and up to about twice thevalue obtained with naloxone as control.

Naltrexone (NTX) can also be used as a control inhibitor. Averageinhibition values obtained using NTX rather than NLX tend to be 1 or 2percent lower in absolute value than those obtained with NLX. Thus, forexample, where an average inhibition value at a particular concentrationof NLX is 40 percent, one can expect values obtained with NTX to beabout 38 or 39 percent. The binding inhibition values for a contemplatedcompound are determined taking the expected NLX/NTX value differenceinto account.

In other binding studies, U.S. Pat. No. 7,560,882 and No. 8,153,795 toSundermann et al. teach that compounds similar to those of the C-1 andC-2 Series of compounds are useful for the inhibition of the reuptake ofserotonin, noradrenalin reuptake, and have a high affinity forbatrachotoxin (BTX) receptors and/or cannabinoid receptors (CB2). Theresults shown in FIG. 45 illustrate that compounds of Series D that arewithin the definition provided by Sundermann et al. U.S. Pat. No.7,560,882 also inhibit tau hyperphosphorylation and are useful in acontemplated method.

Representative compounds from the present Series A, Series C-1 andSeries C-2 groups were examined by Ricerca Biosciences LLC of Taipei,Taiwan, in competitive binding assay studies using published techniquesto determine whether the compounds could competitively inhibit bindingto any of more than 65 receptors, channels and transporters includingadrenergic receptors to which noradrenalin binds, serotonin receptors,muscarinic receptors to which BTX binds and cannabinoid receptors. Thestudied compounds each exhibited no significant inhibition in each ofthose assays.

Pharmacophore Determinations

One aspect of the invention is the use of a compound that binds to thepentapeptide of SEQ ID NO: 1 that is present in FLNA to inhibitphosphorylation of the tau protein. In this aspect, the structures ofthe compounds that effectively bind to a pentapeptide of SEQ ID NO: 1 isquite varied but can be unified through the computer-assistedcalculation of a group of pharmacophores shared by those compounds thatso bind.

A contemplated compound useful in a method of the invention contains atleast four of the six pharmacophores of FIGS. 35-40. In preferredpractice, a contemplated compound contains five of the sixpharmacophores of those figures, and more preferably, a contemplatedcompound contains all six of the pharmacophores. Aside from NLX, NTX,methadone, fentanyl, nalorphine, nalbuphine and buprenorphine that bindwell to the FLNA pentapeptide of SEQ ID NO: 1 (VAKGL), compounds of fourstructural series discussed hereinafter are particularly preferred.

An ensemble pharmacophore model was prepared with a programmed generalpurpose computer using the three-dimensional conformations of compoundsin the training sets. Using 0.1 μM data from Example 1 as a startingpoint, 153 compounds out of the list of compounds in the tables ofExample 1 have a binding activity to the FLNA pentapeptide that is lessthan the mean value of 45.54 percent. A “poor binding” compound or “poorbinder” is defined as a compound whose binding inhibition is equal to orless than the mean value of 45.54 percent in an assay as conducted inExample 1, whose results are shown in the tables of Example 1. Thetraining set consists of ten compounds known to bind to the FLNApentapeptide, the above poor binding 153 compounds and also about 1000random compounds selected from ZINC database at zinc.docking.org.

The selection of pharmacophores involves in the following steps: 1)Three-dimensional computer-generated conformations of all compounds werefirst prepared. 2) A set of 4-point pharmacophores present in most ofknown active compounds was derived. 3) Using known inactive and randomselected compounds as reference compounds, only those pharmacophoresthat were not present in the most of the reference compounds wereidentified as relevant to FLNA binding activity. 4) Six 4-pointpharmacophores were finally identified from those determined above tobest represent the 10 active compounds.

An untested compound that contains four out of the six pharmacophoreshas about a 20 percent chance to be an active binder in FLNApentapeptide. A compound containing five of the six pharmacophores hasabout a 32 percent chance to be an active binder in FLNA pentapeptide,and about a 60 percent chance when containing six of the sixpharmacophores.

The Molecular Operating Environment (MOE) software from ChemicalComputing Group, Montreal, Quebec, Canada, was used to program a generalpurpose computer to generate three-dimensional conformations, to derive4-point pharmacophores from active compounds, and to test thesepharmacophores against known inactive compounds and random selectedcompounds. Pharmacophore modeling as used herein is carried out asdiscussed and explained in Penzotti et al., J Med Chem, 2002,45(9):1737-1740 (2002); Siew et al., Bioorg Med Chem Lett,21(10):2898-2905 (15 May 2011); Leong, Chem Res Toxicol, 20(2):217-226(2007); and Lin, chemcomp.com/journal/ph4.htm.

In some embodiments, it is preferred that a FLNA-binding compound alsobe a MOR agonist. In other embodiments, it is preferred that theFLNA-binding compound not be a MOR agonist. A compound is defined hereinas not being a MOR agonist if it has less than about 80 percent thebinding of [D-Ala2,N-MePhe4,Gly-ol]-enkephalin (DAMGO) at either of thetwo concentrations used in the Table of Example 2.

The ten known FLNA pentapeptide-binding training set compounds are shownbelow along with their alpha-numeric designations used herein. Of the

above ten compounds used in the training set for determining thepharmacophores, nine contained all six pharmacophores. Naloxonecontained five of the six. Examining several more of the structures ofthe four groups of compounds (Series A, Series B, Series C-1 and SeriesC-2) shown in the tables and assayed in Example 1 hereinafter, twentyfurther compounds contained to five of the six pharmacophores, andanother twenty contained four of the six.

Specifically Contemplated FLNA-Binding Compounds

A compound contemplated for use in a contemplated method binds to theFLNA pentapeptide of SEQ ID NO: 1, and contains at least four of the sixpharmacophores of FIGS. 35-40. Such a compound can have a variedstructure as noted before. Regardless of that structural variance, acontemplated compound inhibits the binding of labeled naloxone(FITC-NLX) to the biotinylated-VAKGL pentapeptide (Bn-VAKGL; SEQ IDNO: 1) bound to coated streptavidin plates to an extent that is at leastabout 80 percent of the value obtained when using naloxone as aninhibitor at the same concentration and under conditions definedhereinafter in Example 1, and can be about twice the value for naloxoneat the same concentration.

Compounds having four exemplary structures have been found to bind wellto the pentapeptide of SEQ ID NO: 1. Those compounds are referred toherein as Series A, Series B, Series C-1, Series C-2, Series D andSeries E. Inhibition of tau phosphorylation by Compounds A, B and C andSeries D are illustrated herein and those compounds are representativeof those structural series. Compounds of Series E overlap with those ofSeries C-1 and -2 and are therefore also included herein. The generalstructures of the compounds of each series are shown below, followed bymore specific disclosures.

A pharmaceutically acceptable salt of a compound of each of the aboveFormulas is also contemplated. A compound having an asymmetrical(chiral) carbon or a salt of such a compound can exist in the form ofstereoisomers, that are two enantiomers. The invention relates both toeach enantiomer separately, and to their mixture; i.e., to bothenantiomeric forms (d and l, or R and S) and to their mixture.Additionally, where two or more chiral centers are present,stereoisomers called diastereomers can form, and diastereomers are alsocontemplated.

As will be seen from the following definitions, a contemplated compoundcan contain one or more deuterated carbon atoms, in which deuterium isdesignated by its usual chemical designation, D. Deuterated compoundscan be useful in studying the mechanism of drug interactions with livingorganisms for the elucidation of metabolic and biosynthetic pathways.Deuteration can also extend the half-life of a contemplated compound invivo because a carbon-deuterium (C-D) bond is stronger than aCarbon-hydrogen (C—H) bond thereby requiring more energy input for bondcleavage. See, Blake et al., 1975 J. Pharm. Sci. 64(3):367-391; andNelson et al., 2003 Drug Metab. Dispos. 31(12):1481-1498, and thecitations therein. Contemplated deuterated compounds are prepared usingwell-known reactions.

More particularly, a compound of Series A corresponds in structure toFormula A, below,

wherein

R¹ and R² are the same or different and are independently H, halogen,C₁-C₁₂ hydrocarbyl, C₁-C₆ acyl, C₁-C₆ hydrocarbyloxy, CF₃ and NR³R⁴,wherein R³ and R⁴ are the same or different and are H, C₁-C₄hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R³ and R⁴together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur;

A and B are the same or different and are CH₂, CDH or CD₂ (where D isdeuterium);

X is OH or NR⁵R⁶ wherein R⁵ and R⁶ are the same or different and are H,C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵ and R⁶together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur;

NR⁷R⁸, R⁷ and R⁸ are the same or different and are H, C₁-C₆ hydrocarbyl,C₁-C₆ acyl, C₁-C₆ hydrocarbylsulfonyl, or R⁷ and R⁸ together with thedepicted nitrogen form a ring structure W;

W contains 5 to 14 atoms in the ring structure including the depictednitrogen, and preferably up to 12 atoms. W can optionally contain: a) 1or 2 further hetero atoms that are independently oxygen, nitrogen orsulfur, and b) one or more substituent groups bonded to one or more ringatoms, in which the one or more substituents contain a total of up to 8atoms, and preferably up to 6 atoms, selected from the group consistingof carbon, nitrogen, oxygen and sulfur, and mixtures thereof.

A dashed line (- - - -) represents an optional double bond.

In regard to a contemplated compound, R¹ and R² are preferably otherthan methyl and isopropyl, respectively, when W is N-morpholinyl ordimethyl-N-morpholinyl and the optional double bonds are absent.

A preferred compound of Formula A is a compound of Formula I, below, inwhich A, B, X, W and R¹ and R² are as defined above.

In one preferred embodiment, a contemplated compound corresponds instructure to Formula Ia

Here, R¹ and R² are the same or different and are independently H, orC₁-C₆ hydrocarbyl; A and B are the same or different and are CH₂, CDH orCD₂; W is a ring structure that contains 5 to 14 atoms in the ringstructure including the depicted nitrogen, and can optionally contain:a) 1, 2 or 3 further hetero atoms that are independently oxygen,nitrogen or sulfur, and b) one or more substituent groups bonded to oneor more ring atoms, in which the one or more substituent contain a totalof up to 14 atoms, preferably up to 12 atoms and more preferably up to 8atoms selected from the group consisting of carbon, nitrogen, oxygen andsulfur, and mixtures thereof. The dashed line (- - - -) represents 1, 2,or 3 optional double bonds. Preferably, R¹ and R² are other than methyland isopropyl, respectively, when W is N-morpholinyl ordimethyl-N-morpholinyl, and the optional double bonds are absent.

In preferred practice for some embodiments of a compound of eitherFormula I or Formula Ia, W further includes one or more substituentgroups bonded to one or more ring atoms, in which those one or moresubstituents contain a total of up to 8 atoms selected from the groupconsisting of carbon, nitrogen, oxygen and sulfur, and mixtures thereof.Hydrogen atoms bonded to those atoms are not counted.

In one preferred embodiment, a compound of Formulas I and Ia has thestructure of Formula II, whereas in another preferred embodiment, acompound of Formulas I and Ia has the structure of a compound of FormulaIII.

In a compound of both of Formulas II and III, A, B, W and X are aspreviously defined for a compound of Formulas I and Ia, above. R¹ and R²for a compound of Formula II are defined as R¹ and R² for a compound ofFormula Ia, whereas R¹ and R² for a compound of Formula III are definedas R¹ and R² for a compound of Formula I.

More preferably, the R¹ and R² groups of a compound of Formula IIcontain 3 to 5 carbon atoms. For some compounds of Formula III, R¹ is Hand R² is halogen, C₁-C₆ hydrocarbyl, C₁-C₆ acyl, C₁-C₆ hydrocarbyloxyor NR³R⁴, whereas for others, both R groups are other than H, but chosenas defined above.

In a compound of either Formula II or Formula III, W can optionallycontain 1 or 2 further hetero atoms that are independently oxygen,nitrogen or sulfur, and more preferably still, contains at least onesuch hetero atom. It is also preferred that W further includes one ormore substituent groups bonded to one or more ring atoms, in which theone or more substituents contain a total of up to 8 atoms selected fromthe group consisting of carbon, nitrogen, oxygen and sulfur, andmixtures thereof, and hydrogens bonded to those atoms are not counted.

A particularly preferred compound of Formulas II and III has a structureof Formulas IIa and IIIa, wherein the other groups A, B, W, R¹ and R²are as defined above.

A compound of Series B corresponds generally to the Formula I, below

wherein

n=0 or 1;

m=0 or 1;

m+n=0, 1 or 2;

W is an aromatic ring containing 0, 1 or 2 hetero atoms that can benitrogen, oxygen or sulfur, or mixtures thereof in the ring;

R¹ is selected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, halogen, cyano, C₁-C₆ hydrocarbyloxyhydrocarboxylene,trifluoromethyl, and hydroxyl;

R² is selected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, C₁-C₆ hydrocarbyloxyhydrocarboxylene and halogen;

R³ is absent or C₁-C₆ hydrocarbyl;

R⁴ is C₁-C₆ hydrocarbyl;

X⁻=an anion or is absent when R³ is absent;

the dashed line indicates an optional double bond between the depictedcarbon atoms; and

the wavy line indicates that the depicted phenyl substituent can be inthe Z or E configuration when the optional double bond is present.

Illustrative anions can be monovalent or polyvalent. A contemplatedanion is pharmaceutically acceptable and includes phosphate, hydrogenphosphate, dihydrogenphosphate, sulfate, bisulfate, chloride, bromide,iodide, acetate, formate, benzenesulfonate, methanesulfonate,toluenesulfonate and the like as are well known. These and other anionsare listed in Berge et al., 1977 J. Pharm Sci. 68(1):1-19.

It is preferred that m+n=1 or 2, and the optional double bond is absent,and is rather a saturated, single bond.

In preferred practice, W is a six-membered ring, although five memberedrings are also contemplated. Thus, a contemplated aromatic ring that caninclude zero, one or two hetero atoms that are nitrogen, oxygen orsulfur or mixtures thereof include phenyl, pyridyl, furanyl, imidazyl,oxazolyl and the like. In some preferred embodiments, W is free of (haszero) ring nitrogen atoms. In other embodiments, preferred compoundshave W groups that are free of ring hetero atoms, having only ringcarbon atoms.

W preferably further includes one or more substituent groups (R¹ and R²)to one or more ring atoms, in which those one or more substituentscontain a total of up to 12 atoms selected from the group consisting ofcarbon, nitrogen, oxygen and sulfur, and mixtures thereof, with hydrogenatoms not being counted. Preferred substituent groups on ring W have anoxygen atom bonded to the W ring. Such compounds are preferably C₁-C₆hydrocarbyloxy groups such as methoxy groups.

The Z-containing group can be a keto group or can be reduced to ahydroxyl group. Both groups are preferred.

In some embodiments, both R³ and R⁴ are C₁-C₆ hydrocarbyl groups thatare both methyl. In other embodiments, one is an ethyl group and theother is methyl or absent. When R³ is absent, a Series B compound is atertiary amine.

In one preferred embodiment, a Series B compound of Formula I has thestructure of Formula II,

wherein

n=0 or 1;

m=0 or 1;

m+n=0, 1 or 2;

X⁻=an anion;

R¹ is selected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, halogen, cyano, C₁-C₆ hydrocarbyloxyhydrocarboxylene,trifluoromethyl, and hydroxyl;

R² is selected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, C₁-C₆ hydrocarbyloxyhydrocarboxylene and halogen;

the dashed line indicates an optional double bond between the depictedcarbon atoms; and

the wavy line indicates that the depicted phenyl substituent can be inthe Z or E configuration when the optional double bond is present.

In some preferred embodiments, R²═H. In some such embodiments, R¹includes an oxygen atom bonded to the depicted phenyl ring, and thatoxygen is preferably part of a C₁-C₆ hydrocarbyloxy group. For maycompounds, it is preferred that

In yet other preferred embodiments, a contemplated Series B compound ofhas a structure that corresponds to Formula III, below

here,

n=0 or 1;

m=0 or 1;

m+n=0, 1 or 2;

X⁻=an anion;

R¹ is selected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, halogen, cyano, C₁-C₆ hydrocarbyloxyhydrocarboxylene,trifluoromethyl, and hydroxyl; and

R² is selected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, C₁-C₆ hydrocarbyloxyhydrocarboxylene and halogen.

As was the case for other Series B compound embodiments, R² is sometimesH, and one or both of R¹ and R² are C₁-C₆ hydrocarbyloxy groups such asmethoxy. A pharmaceutically acceptable salt of a compound of Formula I,II and III and all of the remaining Series B formulas disclosed hereinis also contemplated.

A compound of Series C-1 corresponds generally to the Formula A, below

In Formula Series C-1 Formula A, G and W are selected from the groupconsisting of NR²⁰, NR⁷, CH₂, S and O, where R⁷ is H, C₁-C₁₂hydrocarbyl, or C₁-C₁₂ hydrocarboyl (acyl) and R²⁰ is a group X-circleA-R¹ as defined hereinafter, and G and W are preferably NR²⁰ and NR⁷. Inone preferred embodiment, only one of G and W is NR⁷ and one of G and Wmust be NR⁷ or NR²⁰;

X and Y are the same or different and are SO₂, C(O), CH₂, CD₂ (where Dis deuterium), OC(O), NHC(NH), NHC(S) or NHC(O);

Q is CHR⁹ or C(O); Z is CHR¹⁰ or C(O); each of d, e, f and k is eitherzero or one and the sum of (d+e+f+k)=2. In some embodiments, e is zerowhen d is zero, and g is zero when f is zero. In other embodiments, d iszero when f is zero, or e is zero when g is zero.

Each of m, n and p is zero or one and the sum of m+n+p is 2 or 3 for allembodiments. Each of m and n is preferably 1, and p is preferably zeroso that the sum of m+n+p is preferably 2.

The circles A and B are the same or different aromatic or heteroaromaticring systems. Groups R¹ and R² are the same or different and each can behydrogen or represent up to three substituents other than hydrogen thatthemselves can be the same or different; i.e., R^(1a), R^(1b), andR^(1c), and R^(2a), R^(2b), and R^(2c). Each of those six groups,R^(1a-c) and R^(2a-c), is separately selected from the group consistingof H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆hydrocarbyloxycarbonyl, trifluoromethyl, trifluoromethoxy, C₁-C₇hydrocarboyl (acyl), hydroxy-, trifluoromethyl- (—CF₃) orhalogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₆hydrocarbyloxysulfonyl, halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇hydrocarbyl carboxylate [C(O)O—C₁-C₇ hydrocarbyl], carboxamide[C(O)NR³R⁴] or sulfonamide [S(O)₂NR³R⁴] wherein the amido nitrogen ineither group has the formula NR³R⁴ wherein R³ and R⁴ are the same ordifferent and are H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with thedepicted nitrogen form a 5-7-membered ring that optionally contains 1 or2 additional hetero atoms that independently are nitrogen, oxygen orsulfur,

MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringed aryl groupas described previously, and NR⁵R⁶, wherein R⁵ and R⁶ are the same ordifferent and are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄hydrocarbylsulfonyl, or R⁵ and R⁶ together with the depicted nitrogenform a 5-7-membered ring that optionally contains 1 or 2 additionalhetero atoms that independently are nitrogen, oxygen or sulfur;

R⁸, R⁹, and R¹⁰ are each H, or two of R⁸, R⁹, and R¹⁰ are H and one is aC₁-C₈ hydrocarbyl group that is unsubstituted or is substituted with upto three atoms that are the same or different and are oxygen or nitrogenatoms;

R¹¹, R¹², R¹³ and R¹⁴ are all H, or one of the pair R¹¹ and R¹² or thepair R¹³ and R¹⁴ together with the depicted ring form a saturated orunsaturated 6-membered ring, and the other pair are each H, or they areH and D as recited herein (in this subparagraph).

Also in the above preferred embodiment, R¹ and R² are not both methoxywhen X and Y are both SO₂, W is O and p is zero.

In another preferred embodiment,

i) only one of G and W is NR²⁰,

ii) one of G and W must be NR²⁰,

iii) one of G and W is other than NR⁷ in which R⁷ is H or an aliphaticC₁ hydrocarbyl; i.e., methyl, when (a) the sum of m+n+p is 2, and (b)the other of G and W is NR²⁰ bonded to a Z or Q, respectively, that isC(O), and

iv) when X and Y are both SO₂, W is O, Q is CH₂, p is zero, and d and fare both 1, R¹ and R² are other than (a) both H, methoxy, orC₁-C₃-hydrocarbyl, (b) H, halogen and C₁-C₃-hydrocarbyl, (c) H andC₁-C₃-hydrocarbyl, (d) halogen and C₁-C₃-hydrocarbyl, or (e) H andhalogen.

R¹ and R² are preferably also not both methoxy when X and Y are bothSO₂, W is O and p is zero in the above-preferred embodiment.

A pharmaceutically acceptable salt of a compound of Series C-1 Formula Aand all of the remaining Series C-1 formulas disclosed herein is alsocontemplated.

In one preferred Series C-1 embodiment, e and g are both zero and acompound of Series C-1 Formula A becomes a compound of Series C-1Formula B, below

In Formula B, the letters of the formula G, J, E, F, K, W, Q, Z, d, e,f, k, n, m, p, X, Y, circle A and circle B and all R groups are aspreviously defined for a compound of Formula A of Series C-1.Preferably, R¹ and R² are not both methoxy when X and Y are both SO₂, Wis O and p is zero.

In all of the following sub-generic formulas of a compound of SeriesC-1, the formula letters of G, J, E, F, K, W, Q, Z, d, e, f, k, n, m, p,X, Y, circle A and circle B and all R groups are as previously definedfor a compound of Formula A of Series C-1, unless otherwise defined.Additionally, the previously stated preferences also apply unless adepicted structural formula precludes such a preference.

More preferably, a compound of Series C-1 Formula B corresponds instructure to Series C-1 Formula I, below

In Series C-1 Formula I, X and Y are the same or different and are SO₂,C(O), CH₂, CD₂, NHC(NH), OC(O), NHC(S) or NHC(O);

W is NR⁷, CH₂, S or O, where R⁷ is H, C₁-C₁₂ hydrocarbyl, or C₁-C₁₂hydrocarboyl (acyl), and is preferably NR⁷;

Q is CHR⁹ or C(O);

Z is CHR¹⁰ or C(O);

J and F are the same or different and are CH or CD (where D isdeuterium);

each of m, n and p is zero or one and the sum of m+n+p is 2 or 3,preferably 2; and

the circles A and B are the same or different aromatic or heteroaromaticring systems that contain one ring or two fused rings. Groups R¹ and R²are the same or different and each can be hydrogen or represent up tothree substituents other than hydrogen that themselves can be the sameor different; i.e., R^(1a), R^(1b), and R^(1c), and R^(2a), R^(2b), andR^(2c). Each of those six groups, R^(1a-c) and R^(2a-c), is separatelyselected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, trifluoromethyl, trifluoromethoxy, C₁-C₇ hydrocarboyl(acyl), hydroxy-, trifluoromethyl- (—CF₃) or halogen-substituted C₁-C₇hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, halogen (F, Cl or Br, andpreferably Cl), nitro, phenyl, cyano, carboxyl, C₁-C₇ hydrocarbylcarboxylate [C(O)O—C₁-C₇ hydrocarbyl], carboxamide [C(O)NR³, R⁴] orsulfonamide [SO₂NR³R⁴] wherein the amido nitrogen of either group (thecarboxamide or sulfonamide) has the formula NR³R⁴ wherein R³ and R⁴ arethe same or different and are H, C₁-C₄ hydrocarbyl, or R³ and R⁴together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur, MAr, where M is where M is —CH₂—, —O— or—N═N— and Ar is a single-ringed aryl group, and NR⁵R⁶ wherein R⁵ and R⁶are the same or different and are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl,C₁-C₄ hydrocarbylsulfonyl, or R⁵ and R⁶ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur;

R⁸, R⁹, and R¹⁰ are each H, or two of R⁸, R⁹, and R¹⁰ are H and one is aC₁-C₈ hydrocarbyl group that is unsubstituted or is substituted with upto three atoms that are the same or different and are oxygen or nitrogenatoms; and

R¹¹, R¹², R¹³ and R¹⁴ are all H, or R¹¹ and R¹³ are H and R¹² and R¹⁴are H or D, or one of the pair R¹¹ and R¹² or the pair R¹³ and R¹⁴together with the depicted ring form a saturated or unsaturated6-membered ring, and the other pair are each H or they are H and D asrecited herein (in this subparagraph).

Preferably, R¹ and R² are not both methoxy when X and Y are both SO₂, Wis O and p is zero.

In other preferred embodiments, X and Y are the same. X and Y arepreferably both C(O) or both SO₂, and more preferably are both SO₂. Inthose and other embodiments, W is preferably O. It is also preferredthat p be zero.

A contemplated aromatic or heteroaromatic ring system of circle A orcircle B can contain one ring or two fused rings, and preferablycontains a single aromatic ring. An illustrative aromatic orheteroaromatic ring system is selected from the group consisting ofphenyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl(1,3,5-triazinyl, 1,2,4-triazinyl and 1,2,3-triazinyl), furanyl,thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, naphthyl,benzofuranyl, isobenzofuranyl, benzothiophenyl, isobenzothiophenyl,benzoxazolyl, benzisoxazole, quinolyl, isoquinolyl, quinazolyl,cinnolinyl, quinoxalinyl, naphthyridinyl, benzopyrimidinyl, and mixturesthereof. The mixtures of the previous sentence occur when circle A andcircle B aromatic or heteroaromatic ring systems are different.

An illustrative single-ringed aryl group of substituent MAr is selectedfrom the group consisting of phenyl, pyridyl, pyrazinyl, pyrimidinyl,pyridazinyl, triazinyl (1,3,5-triazinyl, 1,2,4-triazinyl and1,2,3-triazinyl), furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl andisothiazolyl.

Phenyl is a preferred aromatic or heteroaromatic ring system of circle Aand circle B. Phenyl, pyridinyl and furanyl are preferred single-ringedaryl groups, Ar, of a MAr substituent, with phenyl being particularlypreferred.

There are several independent and separate preferences regarding thesubstituent R groups. Thus, R¹ and R² are preferably the same singlesubstituent other than hydrogen, so that circle A and circle B bothcontain a single substituent other than hydrogen. The single substituentof R¹ and R² is preferably located at the same relative position intheir respective ring systems.

Thus, X and Y can form a sulfonamido, a carboxamido, a urea, a thiourea,a guanidino or methylene linkage from the circle A or circle B ringsystem to a depicted nitrogen atom of the central spiro ring. A compoundhaving a central ring that is a spiro 6,6-ring system or a spiro5,6-ring system, along with one nitrogen and one oxygen or two nitrogenatoms is contemplated. Illustrative central spiro rings are shown belowwhere wavy lines are used to indicate the presence of covalent bonds toother entities, and where R⁷ is defined above and R⁸ is H.

Illustrative compounds of Formula A in which d and e are each zero andR¹¹, R¹² and R¹³ are each H have asymmetric Spiro ring structures a fewof which are shown below with wavy lines indicating the presence ofcovalent bonds to other entities, and R⁷ is defined above and R⁸ is H.

In preferred practice, p is zero, e and g are both zero and R¹¹, R¹² andR¹³ are all H, so the central ring is a spiro 5,6-ring system whose6-membered ring is unsubstituted and in which the spiro bonds are in the4-position relative to the nitrogen of the 6-membered ring. It isseparately preferred that W be O. A compound in which X and Y are thesame is preferred. It is also separately preferred that X and Y both beSO₂ (sulfonyl).

A particularly preferred compound of Series C-1 Formula A that embodiesthe above separate preferences is a compound of Series C-1 Formula II

wherein

circle A and circle B, Z, Q, m, n, p, R¹, R² and R⁸ are as describedabove for a compound of Series C-1, unless the formula as shownprecludes a definition provided for a compound of Formula A; and J and Fare the same or different and are CH₂, CHD or CD₂ (where D isdeuterium).

It is more preferred that circle A and circle B are each phenyl, furanylor pyridyl and R¹ and R² is each a single substituent. There are severalindependent and separate preferences regarding the substituent R groups.Thus, R¹ and R² are preferably the same. R¹ and R² are also preferablylocated at the same relative position in their respective rings. Thus,if R¹ is 4-cyano, R² is also 4-cyano. It is also preferred that the sumof m+n+p=2 so that the upper depicted ring contains 5-ring atoms.

Preferred R¹ and R² substituent groups do not themselves provide apositive or negative charge to a compound at a pH value of about7.2-7.4.

In other embodiments, a particularly preferred compound of Series C-1Formula A is a compound of Series C-1 Formula III

wherein

circle A and circle B, Z, Q, m, n, p, R¹, R² and R⁸ are as describedpreviously for a compound of Series C-1 unless the formula as shownprecludes a prior definition; J and F are the same or different and areCH₂, CHD or CD₂ (where D is deuterium); and X and Y are both CO, or Xand Y are different and are SO₂, C(O), CH₂, CD₂ (where D is deuterium),OC(O), NHC(NH), NHC(S) or NHC(O). Previous preferences are alsoapplicable unless precluded by the above structural formula.

More preferably, circle A and circle B are each phenyl, furanyl orpyridyl. R¹ and R² are the same and are selected from the groupconsisting of trifluoromethyl, C₁-C₆ acyl, C₁-C₄ alkylsulfonyl, halogen,nitro, cyano, carboxyl, C₁-C₄ alkyl carboxylate, carboxamide wherein theamido nitrogen has the formula NR³R⁴ wherein R³ and R⁴ are the same ordifferent and are H, C₁-C₄ alkyl, and NR⁵R⁶ wherein R⁵ and R⁶ are thesame or different and are H, C₁-C₄ alkyl, C₁-C₄ acyl, C₁-C₄alkylsulfonyl.

It is still more preferred that R¹ and R² each be a single substituent.There are several independent and separate preferences regarding thesubstituent R groups. R¹ and R² are preferably the same. R¹ and R² arealso preferably located at the same relative position in theirrespective rings. Thus, if R¹ is 4-cyano, R² is also 4-cyano. It is alsopreferred that p=0, and that the sum of m+n+p=2, so that the upperdepicted ring contains 5-ring atoms.

In still further embodiments, a particularly preferred compound ofSeries C-1 Formula A is a compound of Series C-1 Formula IV

wherein

circle A and circle B, Z, g, m, n, p, R¹, R², R⁷ and R⁸ are as describedpreviously for a compound of Series C-1 unless the formula as shownprecludes such a prior definition; J and F are the same or different andare CH₂, CHD or CD₂ (where D is deuterium); and X and Y are the same ordifferent and are SO₂, C(O), CH₂, CD₂ (where D is deuterium), OC(O),NHC(NH), NHC(S) or NHC(O). Previous preferences are also applicableunless precluded by the above structural formula.

More preferably, circle A and circle B are each phenyl, furanyl orpyridyl. R¹ and R² are the same and are selected from the groupconsisting of trifluoromethyl, C₁-C₆ acyl, C₁-C₄ alkylsulfonyl, halogen,nitro, cyano, carboxyl, C₁-C₄ alkyl carboxylate, carboxamide wherein theamido nitrogen has the formula NR³R⁴ wherein R³ and R⁴ are the same ordifferent and are H, C₁-C₄ alkyl, and NR⁵R⁶ wherein R⁵ and R⁶ are thesame or different and are H, C₁-C₄ alkyl, C₁-C₄ acyl, C₁-C₄alkylsulfonyl.

It is still more preferred that R¹ and R² each be a single substituent.There are several independent and separate preferences regarding thesubstituent R groups. R¹ and R² are preferably the same. R¹ and R² arealso preferably located at the same relative position in theirrespective rings. Thus, if R¹ is 4-cyano, R² is also 4-cyano. It is alsopreferred that the sum of m+n=1, so that the upper depicted ringcontains 5-ring atoms.

It is noted that the previously mentioned preferences regarding E, J, F,G, K, Q, W, X, Y, Z, d, e, f, k, n, m, p, circle A and circle B, and allof the R groups as are appropriate for a particular formula apply to acompound of Series C-1 Formulas A, B, and I-IV.

A compound of Series C-2 corresponds generally to the Formula A, below

In Series C-2 Formula A,

Q is CHR⁹ or C(O), Z is CHR¹⁰ or C(O), and only one of Q and Z is C(O);

each of m and n and p is zero or one and the sum of m+n+p is 2 or 3,preferably 2;

each of G, P and W is selected from the group consisting of NR²⁰, NR²,NR⁷, S and O, where R⁷ and R² are the same or different and are H,C(H)_(v)(D)_(h) where each of v and h is 0, 1, 2 or 3 and v+h=3,C(H)_(q)(D)_(r)-aliphatic C₁-C₁₁ hydrocarbyl where each of q and r is 0,1, or 2 and q+r=0, 1 or 2, (including aliphatic C₁-C₁₂ hydrocarbyl whenq+r=0), aliphatic C₁-C₁₂ hydrocarbyl sulfonyl or aliphatic C₁-C₁₂hydrocarboyl (acyl), and R²⁰ is X-circle A-R¹ as defined hereinafter.

Preferably, in one embodiment,

i) only one of G, P and W is NR²⁰,

ii) one of G, P and W must be NR²⁰,

iii) P is NR² when other than NR²⁰,

iv) one of G and W is other than NR² or NR⁷ in which R² and R⁷ is H oran aliphatic C₁ hydrocarbyl when (a) the sum of m+n+p is 2 and (b) theother of G and W is NR²⁰, NR², or NR⁷ bonded to a Z or Q, respectively,that is C(O), and

v) P is NR² in which R² is other than —S(O)₂C₁-C₃-hydrocarbyl when (a)the sum of m+n+p is 2 and the Q or Z present is CH₂, (b) the G or W thatis not NR²⁰ is O, and (c) R²⁰ is —S(O)₂-phenyl-R¹, where R¹ is H,C₁-C₃-hydrocarbyl or halogen.

Each of d, e, f and k is either zero or one and the sum of (d+e+f+k)=2.In some embodiments, e is zero when d is zero, and k is zero when f iszero. In other embodiments, e is zero when k is zero, and f is zero whend is zero.

J and F are the same or different and are CH or CD (where D isdeuterium).

E and K are the same or different and are CH₂, CHD or CD₂ (where D isdeuterium).

X is SO₂, C(O), CH₂, CD₂, OC(O), NHC(NH), NHC(S) or NHC(O), preferablySO₂, C(O) or CH₂. In some embodiments, X is more preferably CH₂ or SO₂.In other embodiments, X is preferably SO₂, NHC(NH), NHC(S) or NHC(O).

Circle A is an aromatic or heteroaromatic ring system that preferablycontains a single ring, but can also contain two fused rings. R¹ is H orrepresents up to three substituents, R^(1a), R^(1b), and R^(1c), thatthemselves can be the same or different, wherein each of those threegroups, R^(1a-c), is separately selected from the group consisting of H,C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆ hydrocarbyloxycarbonyl,trifluoromethyl, trifluoromethoxy, C₁-C₇ hydrocarboyl, hydroxy-,trifluoromethyl- (—CF₃) or halogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl, halogen (F, Cl, orBr, and preferably Cl) nitro, phenyl, cyano, carboxyl, C₁-C₇ hydrocarbylcarboxylate [C(O)O—C₁-C₇ hydrocarbyl], carboxamide [C(O)NR³, R⁴] orsulfonamide [S(O)₂NR³R⁴],

-   -   wherein the amido nitrogen in either amide group has the formula        NR³R⁴ in which R³ and R⁴ are the same or different and are H,        C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depicted        nitrogen form a 5-7-membered ring that optionally contains 1 or        2 additional hetero atoms that independently are nitrogen,        oxygen or sulfur,

MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringed aryl orheteroaryl group and NR⁵R⁶ wherein R⁵ and R⁶ are the same or differentand are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, orR⁵ and R⁶ together with the depicted nitrogen form a 5-7-membered ringthat optionally contains 1 or 2 additional hetero atoms thatindependently are nitrogen, oxygen or sulfur.

R⁸, R⁹, and R¹⁰ are each H, which is preferred, or two of R⁸, R⁹, andR¹⁰ are H and one is a C₁-C₈ hydrocarbyl group that is unsubstituted oris substituted with up to three atoms that are the same or different andare oxygen or nitrogen atoms (including hydrogens as appropriate).

R¹¹, R¹², R¹³ and R¹⁴ are all H, or R¹¹ and R¹³ are H and R¹² and R¹⁴are H or D, or one of the pair R¹¹ and R¹² or the pair R¹³ and R¹⁴together with the depicted ring form a saturated or unsaturated6-membered ring, and the other pair are each H or they are H and D asrecited herein (in this subparagraph).

In another preferred embodiment of a compound of Formula A, above,

i) only one of G, P and W is NR²⁰,

ii) one of G, P and W must be NR²⁰, and

iii) P is NR² when other than NR²⁰.

Additionally, Q is CHR⁹ or C(O); and

Z is CHR¹⁰ or C(O), with the other of J, E, F, K, X, Z, d, e, f, k, n,m, p, circle A, and all of the R groups being defined as discussed aboveunless precluded by the structural formula.

A pharmaceutically acceptable salt of a compound of Series C-2 Formula Aand all of the remaining formulas disclosed herein is also contemplated.

In preferred embodiments, a compound of Series C-2 Formula A correspondsin structure to either Formula B or Formula C, can be present as apharmaceutically acceptable salt, and can optionally be presentincluding both individual enantiomeric forms, a racemate, diastereomersand mixtures thereof.

In a compound of Series C-2 that corresponds in structure to Series C-2Formula B, G and W are selected from the group consisting of NR²⁰, NR⁷,S and O, where R² and R⁷ are the same or different and areC(H)_(v)(D)_(h) (where D is deuterium) and where each of v and h is 0,1, 2 or 3 and v+h 3, C(H)_(q)(D)_(r)-aliphatic C₁-C₁₁ hydrocarbyl (whereD is deuterium) where each of q and r is 0, 1, or 2 and q+r=0, 1 or 2,aliphatic C₁-C₁₂ hydrocarbyl sulfonyl or aliphatic C₁-C₁₂ hydrocarboyl,or R² and R²⁰ are the same or different, and R²⁰ is X-circle A-R¹.

Preferably in one embodiment,

i) only one of G and W is NR²⁰,

ii) one of G and W must be NR²⁰,

iii) the G or W that is not NR²⁰ is other than NR² or NR⁷ in which R² orR⁷ is H or an aliphatic C₁ hydrocarbyl when (a) the sum of m+n+p is 2and (b) the G or W that is NR²⁰ is bonded to a Z or Q, respectively,that is C(O), and

iv) R² of the depicted NR² is other than —S(O)₂C₁-C₃-hydrocarbyl when(a) the sum of m+n+p is 2 and the Q or Z that is present is CH₂, (b) theG or W that is not NR²⁰ is O, and (c) R²⁰ is —S(O)₂phenyl-R¹, where R¹is H, C₁-C₃-hydrocarbyl or halogen.

In another preferred embodiment:

i) only one of G and W is NR²⁰,

ii) one of G and W must be NR²⁰,

iii) the G or W that is not NR²⁰ is NR² or NR⁷ in which R² or R⁷ is H oran aliphatic C₁ hydrocarbyl,

(iv) the sum of m+n+p is 2, and

(v) the G or W that is NR²⁰ is bonded to a Z or Q, respectively, that isC(O).

In yet another preferred embodiment:

i) only one of G and W is NR²⁰,

ii) one of G and W must be NR²⁰,

iii) the G or W that is not NR²⁰ is NR⁷ that is H or an aliphatic C₁hydrocarbyl,

(iv) the sum of m+n+p is 2,

(v) the G or W that is NR²⁰ is bonded to a Z or Q, respectively, that isC(O),

(vi) R² of the depicted NR² is the same or different R²⁰, and

(vii) R²⁰ is X-circle A-R¹.

For a compound of Formula C, G and W are selected from the groupconsisting of NR², NR⁷, S and O, where R² and R⁷ are the same ordifferent and are H, C(H)_(v)(D)_(h) (where D is deuterium) and whereeach of v and h is 0, 1, 2 or 3 and v+h=3, C(H)_(q)(D)_(r)-aliphaticC₁-C₁₁ hydrocarbyl where each of q and r is 0, 1, or 2 and q+r=0, 1 or2, aliphatic C₁-C₁₂ hydrocarbyl sulfonyl or aliphatic C₁-C₁₂hydrocarboyl.

Preferably, in another embodiment:

i) one of G and W must be NR² or NR⁷, and

ii) one of G and W is other than NR² or NR⁷ in which R² or R⁷ is H or analiphatic C₁ hydrocarbyl when (a) the sum of m+n+p is 2 and (b) theother of G and W is NR² or NR⁷ bonded to a Z or Q, respectively, that isC(O).

In both of Series C-2 Formulas B and C, the symbols X, Z, Q, d, e, f, g,n, m, circle A, and all of the R groups not otherwise defined in theparagraphs following their structural formulas are as defined previouslyfor a compound of Series C-2 Formula A unless the formula as shownprecludes a prior definition. The previously noted preferences are alsoas discussed before unless the formula as shown precludes a priorpreference.

In one embodiment, a preferred compound of Series C-2 Formulas A and Bhas the structure of Formula I

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium); and W, X, Z, Q, n, m, p, circle A, R¹, R², R⁸ and the Rgroups therein defined are as described previously for a compound ofSeries C-2 Formula A, unless the formula as shown precludes a priordefinition. Preferably, i) R² of the depicted NR² is other than—S(O)₂C₁-C₃-hydrocarbyl when (a) the sum of m+n+p is 2 and the Q or Zpresent is CH₂, (b) the G or W that is not NR²⁰ is O, and (c) R²⁰ is—S(O)₂-phenyl-R¹, where R¹ is H, C₁-C₃-hydrocarbyl or halogen, and ii) Wis other than NR² or NR⁷ in which R² or R⁷ is H or an aliphatic C₁hydrocarbyl when (a) the sum of m+n+p is 2 and (b) Z is C(O).

In another preferred embodiment where R⁸ is H, one of n and m is zeroand the remaining Z or Q is CH₂, a compound of Series C-2 Formulas A, Band I has the structure of Series C-2 Formula II

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium); and

X, W, circle A, R¹, R² and the R groups therein defined are as describedpreviously for a compound of Series C-2 Formula A, unless the formula asshown precludes a prior definition. Preferably, R² of the depicted NR²is other than —S(O)₂C₁-C₃-hydrocarbyl when W is O, and X-circle A-R¹ is—S(O)₂-phenyl-R¹, where R¹ is H, C₁-C₃-hydrocarbyl or halogen.

In a further preferred embodiment, where R⁸ is H, a compound of SeriesC-2 Formulas A, B and I has the structure of Series C-2 Formula III

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium);

each of m and n is one; and

W, X, Z, Q, circle A, R¹, R² and the R groups therein defined are asdescribed previously for a compound of Series C-2 Formula A, unless theformula as shown precludes a prior definition.

In one preferred embodiment, i) Z is C(O), ii) Q is CH₂, iii) W is NH,and R² is H or C₁-C₁₂ aliphatic straight, branched or cyclichydrocarbyl, iv) X is preferably CH₂, SO₂, NHC(NH), NHC(S) or NHC(O),and more preferably CH₂. In another preferred embodiment, i) one of Zand Q is C(O), and ii) W is other than NR² or NR⁷ in which R² and R⁷ isH or an aliphatic C₁ hydrocarbyl when Z is C(O), and iii) X ispreferably CH₂, SO₂, NHC(NH), NHC(S) or NHC(O).

In a still further preferred embodiment, i) Z is C(O), ii) Q is CH₂,iii) W is NH, (vi) R² is the same or different R²⁰, and (vii) R²⁰ isX-circle A-R¹. In this embodiment, X is preferably CH₂, SO₂, NHC(NH),NHC(S) or NHC(O), more preferably CH₂.

A presently most preferred compound for carrying out a contemplatedmethod corresponds in structure to Formula III, above, in which i) Z isC(O), ii) Q is CH₂, iii) W is NH, and R² is H or a C₁-C₁₂, preferablyC₁-C₈, and more preferably a C₁-C₆, aliphatic straight, branched orcyclic hydrocarbyl group, iv) X is CH₂, and circle A-R¹ is unsubstitutedphenyl so that the substituent X-circle A-R¹ is a benzyl group.Illustrative presently most preferredN-(benzylamido)-unsubstituted-amine compounds include Compounds C0105M,C0115M and C0124M, whose structural formulas are shown below.

In a still further preferred embodiment, a compound of Series C-2Formulas A and C has the structure of Series C-2 Formula IV

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium); and

W, X, Z, Q, circle A, R¹, R², R⁸ and the R groups therein defined are asdescribed previously for a compound of Series C-2 Formula A, unless theformula as shown precludes a prior definition.

In one preferred embodiment, i) W is other than NR² or NR⁷ in which R²or R⁷ is H or an aliphatic C₁ hydrocarbyl, when p is zero and the sum ofm+n+p is 2 and Z is C(O), and ii) R² of the depicted NR² group is otherthan H or an aliphatic C₁ hydrocarbyl, when p is zero and the sum ofm+n+p is 2, W is NR² or NR⁷′ and Q is C(O).

In yet another preferred embodiment where R⁸ is H, one of n and m iszero and the remaining Z or Q is CH₂, a compound of Formulas A, C and IVhas the structure of Series C-2 Formula V

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium); and X, W, circle A, R¹, R² and the R groups thereindefined are as described previously for a compound of Series C-2, unlessthe formula as shown precludes a prior definition.

In still another preferred embodiment, where R⁸ is H, a compound ofSeries C-2 Formulas A, C and I has the structure of Series C-2 FormulaVI

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium); and

each of m and n is one; W, X, Z, Q, circle A, R¹, R² and the R groupstherein defined are as described previously for a compound of SeriesC-2, unless the formula as shown precludes a prior definition.

Preferably, i) one of Z and Q is C(O), ii) W is other than NR² or NR⁷ inwhich R² or R⁷ is H or an aliphatic C₁ hydrocarbyl when Z is C(O), andiii) R² of the depicted NR² group is other than H or an aliphatic C₁hydrocarbyl when W is NR² or NR⁷′ and Q is C(O). In a compound of theabove formula, X is preferably SO₂, NHC(NH), NHC(S) or NHC(O).

It is also noted that the previously mentioned preferences regardingapply to X, W, Z, Q, d, e, f, k, n, m, circle A, and all of the R groupsapply to a compound of Series C-2 Formulas A, B, C, and I-VI, unless theformula as shown precludes a prior definition.

A contemplated aromatic ring (aryl) system of circle A of one of thecontemplated compounds preferably contains a single aromatic ring, butcan also contain two fused aromatic rings. An illustrative circle Aaromatic ring system is selected from the group consisting of phenyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl(1,3,5-triazinyl, 1,2,4-triazinyl and 1,2,3-triazinyl), furanyl,thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, naphthyl,benzofuranyl, isobenzofuranyl, benzothiophenyl, isobenzothiophenyl,benzoxazolyl, benzisoxazole, quinolyl, isoquinolyl, quinazolyl,cinnolinyl, quinoxalinyl, naphthyridinyl, and benzopyrimidinyl.

An illustrative single-ringed aryl or heteroaryl group of a circle Agroup or of a substituent of circle A, MAr, is selected from the groupconsisting of phenyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,triazinyl (1,3,5-triazinyl, 1,2,4-triazinyl and 1,2,3-triazinyl),furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl and isothiazolylgroups.

Phenyl, pyridinyl and furanyl are a preferred aromatic or heteroaromaticring system of circle A, with phenyl being more preferred. Phenyl,pyridinyl and furanyl are also preferred single-ringed aryl orheteroaryl groups, Ar, of a MAr substituent, with phenyl beingparticularly preferred.

From a depicted nitrogen atom of the central spiro rings to the circle Aring system, X and Y can form a sulfonamido (N—SO₂-circle A), acarboxamido [N—C(═O)-circle A), a urea [carbonyldiimino;N—C(═O)—NH-circle A], a thiourea [thiocarbonyldiimino; N—C(═S)—NH-circleA], a guanidino [N—C(═NH)—NH-circle A] or aminomethylene (N—CH₂-circleA) linkage.

Examining a compound of the above Series C-2 formulas more closely, itis seen that that formula defines a double ringed, substituted spirocompound that can have two six-membered rings or one six- and onefive-membered ring, as when one of “m” and “n” is one and the otherzero. One of those rings (the lower ring in the formulas) contains onenitrogen atom in the 6-membered ring and the remaining ring atoms arecarbons. The ring that can contain 5- or 6-ring atoms (upper ring in theformulas) can contain one ring nitrogen and four or five carbons, or twonitrogens, a nitrogen and a sulfur or a nitrogen and an oxygen atomalong with three or four ring carbons. Illustrative central Spiro ringsare shown below where wavy lines are used to indicate the presence ofcovalent bonds to other entities, and where R⁷ is defined above and R⁸is H for clarity.

Illustrative compounds of Series C-2 Formula A in which d and e are eachzero and R¹¹, R¹² and R¹³ are each H have asymmetric spiro ringstructures a few of which are shown below with wavy lines indicating thepresence of covalent bonds to other entities, and R⁷ is defined aboveand R⁸ is again H for clarity.

In preferred practice for the compounds of Series C-2 Formulas A, B andC, p is zero, e and g are both zero and R¹¹, R¹² and R¹³ are all H, sothe central ring is a spiro 5,6-ring system whose 6-membered ring isunsubstituted and in which the spiro bonds are in the 4-positionrelative to the nitrogen of the 6-membered ring. It is separatelypreferred that W be O, S or NR⁷. It is also preferred that X be SO₂(sulfonyl).

The aromatic substituent, the circle A, is linked to one nitrogen atomof the spiro rings by a X group that is SO₂, C(O), CH₂, CD₂, OC(═O),NHC(═NH), NHC(═S) or NHC(═O), preferably SO₂, C(O), CH₂, or CD₂, andmost preferably CH₂ and SO₂. The resulting aromatic substituent isthereby linked to the spiro ring portion by a sulfonamide, an amide, amethylene, a urea, a thiourea or a guanidino linkage. Aryl sulfonamidebridges, aryl amide bridges and phenylmethylene bridges (benzylcompounds) are preferred, with aryl sulfonamide and phenylmethylenebeing particularly preferred.

Many of the compounds of Series A, Series B, Series C-1, Series C-2,Series D and Series E, as well as compounds such as naloxone andnaltrexone not only bind to the peptide of SEQ ID NO: 1, but also bindto MOR and activate or stimulate that receptor. Naloxone and naltrexonebind to MOR about 200 times more poorly than they bind to thepentapeptide of SEQ ID NO: 1. The tables of Example 2 illustraterelative binding abilities of exemplary compounds of Series A, Series B,Series C-1, and Series C-2 based on MOR stimulatory activity.

In some embodiments it is preferred that a compound useful in acontemplated method binds well, to and activates MOR. In those cases, itis preferred that the compound bind to MOR to an extent of at leastabout ±20 percent as well as DAMGO at a concentration shown in thetables, indicating the compound is a complete agonist for the receptor.In other embodiments, it is preferred that a compound useful herein notbind well to MOR. In those embodiments, it is preferred that thecompound exhibit less than about 80 percent the MOR stimulation providedby DAMGO at the same concentration and conditions, down to zerobinding/stimulation. Illustrative binding percentages in the presence ofstated concentrations of DAMGO are illustrated for exemplary compoundsof Series A, Series B, Series C-1, and Series C-2 in the tables ofExample 2, hereinafter.

A 1,4,8-triazaspiro[4,5]-decan-2-one compound of Series D corresponds instructure to the formula

wherein R¹ represents hydrogen; a linear or branched unsubstituted or atleast monosubstituted alkyl group that can comprise at least oneheteroatom as a link; a linear or branched unsubstituted or at leastmonosubstituted alkenyl group that can comprise at least one heteroatomas a link; a linear or branched unsubstituted or at leastmonosubstituted alkynyl group that can comprise at least one heteroatomas a link; an unsubstituted or at least monosubstituted aryl group or anunsubstituted or at least monosubstituted heteroaryl group, which aryland heteroaryl groups may be bonded via a linear or branched alkylenegroup that can comprise at least one heteroatom as a link; or a—C(═O)OR⁷ group that can be bonded via a linear or branched alkylenegroup;

R² represents hydrogen; a linear or branched unsubstituted or at leastmonosubstituted alkyl group that can comprise at least one heteroatom asa link; a linear or branched unsubstituted or at least monosubstitutedalkenyl group that can comprise at least one heteroatom as a link; alinear or branched unsubstituted or at least monosubstituted alkynylgroup that can comprise at least one heteroatom as a link; anunsubstituted or at least monosubstituted aryl group or an unsubstitutedor at least monosubstituted heteroaryl group, which aryl and heteroarylgroup may be bonded via a linear or branched alkylene group that cancomprise at least one heteroatom as a link;

R³ represents a —S(═O)₂—R⁴ group; a —C(═S)NH—R⁵ group; or a —C(═O)NH—R⁶group;

R⁴ represents a —NR¹⁰R¹¹ group; a linear or branched unsubstituted or atleast monosubstituted alkyl group that can comprise at least oneheteroatom as a link; a linear or branched unsubstituted or at leastmonosubstituted alkenyl group that can comprise at least one heteroatomas a link; a linear or branched unsubstituted or at leastmonosubstituted alkynyl group that can comprise at least one heteroatomas a link; an unsubstituted or at least monosubstituted aryl group or anunsubstituted or at least monosubstituted heteroaryl group, which groupsmay be bonded via a linear or branched unsubstituted or at leastmonosubstituted alkylene group that can comprise at least one heteroatomas a link and may be condensed with an unsubstituted or at leastmonosubstituted monocyclic ring system; an unsubstituted or at leastmonosubstituted cycloaliphatic group, that can comprise at least oneheteroatom as a ring member and that can be bonded via a linear orbranched unsubstituted or at least monosubstituted alkylene group thatcan comprise at least one heteroatom as a link and that can be bridgedby a linear or branched unsubstituted or at least monosubstitutedalkylene group;

R⁵ represents a linear or branched unsubstituted or at leastmonosubstituted alkyl group that can comprise at least one heteroatom asa link; a linear or branched unsubstituted or at least monosubstitutedalkenyl group that can comprise at least one heteroatom as a link; alinear or branched unsubstituted or at least monosubstituted alkynylgroup that can comprise at least one heteroatom as a link; anunsubstituted or at least monosubstituted aryl group or an unsubstitutedor at least monosubstituted heteroaryl group, which group may be bondedvia a linear or branched unsubstituted or at least monosubstitutedalkylene group that can comprise at least one heteroatom as a link; anunsubstituted or at least monosubstituted cycloaliphatic group, that cancomprise at least one heteroatom as a ring member or that can be bondedvia a linear or branched unsubstituted or at least monosubstitutedalkylene group that can comprise at least one heteroatom as a link; a—C(═O)OR⁸ group or a —C(═O)OR⁹ group, that can, in either case, bebonded via a linear or branched alkylene group;

R⁶ represents an unsubstituted or at least monosubstituted aryl group oran unsubstituted or at least monosubstituted heteroaryl group, whicharyl and heteroaryl groups may be bonded via a linear or branchedunsubstituted or at least monosubstituted alkylene group that cancomprise at least one heteroatom as a link; or for an unsubstituted orat least monosubstituted cycloaliphatic group, that can comprise atleast one heteroatom as a ring member or that can be bonded via a linearor branched unsubstituted or at least monosubstituted alkylene groupthat can comprise at least one heteroatom as a link;

R⁷, R⁸, R⁹, R¹⁰, and R¹¹, each independently represent a linear orbranched alkyl group, a linear or branched alkenyl group, or a linear orbranched alkynyl group, or a physiologically acceptable salt thereof.

Preferably for a 1,4,8-triazaspiro[4,5]-decan-2-one compoundcorresponding to the formula above, R¹ represents hydrogen; a linear orbranched unsubstituted or at least monosubstituted C₁₋₁₀ alkyl groupthat can comprise at least one heteroatom as a link; a linear orbranched unsubstituted or at least monosubstituted C₂₋₁₀ alkenyl groupthat can comprise at least one heteroatom as a link; a linear orbranched unsubstituted or at least monosubstituted C₂₋₁₀ alkynyl groupthat can comprise at least one heteroatom as a link; an unsubstituted orat least monosubstituted five-membered to fourteen-membered aryl groupor heteroaryl group, that can be bonded via a linear or branched C₁₋₅alkylene group that can comprise at least one heteroatom as a link; a—C═O)OR⁷ group that can be bonded via a linear or branched C₁₋₅ alkylenegroup;

R² represents hydrogen; a linear or branched unsubstituted or at leastmonosubstituted C₁₋₁₀ alkyl group that can comprise at least oneheteroatom as a link; a linear or branched unsubstituted or at leastmonosubstituted C₂₋₁₀ alkenyl group that can comprise at least oneheteroatom as a link; a linear or branched unsubstituted or at leastmonosubstituted C₂₋₁₀ alkynyl group that can comprise at least oneheteroatom as a link; an unsubstituted or at least monosubstitutedfive-membered to fourteen-membered aryl or heteroaryl group, that can bebonded via a linear or branched C₁₋₅ alkylene group that can comprise atleast one heteroatom as a link;

R⁴ represents an NR¹⁰R¹¹ group; a linear or branched unsubstituted or atleast monosubstituted C₁₋₁₀ alkyl group that can comprise at least oneheteroatom as a link; a linear or branched unsubstituted or at leastmonosubstituted C₂₋₁₀ alkenyl group that can comprise at least oneheteroatom as a link; a linear or branched unsubstituted or at leastmonosubstituted C₂₋₁₀ alkynyl group that can comprise at least oneheteroatom as a link; an unsubstituted or at least monosubstitutedfive-membered to fourteen-membered aryl group or heteroaryl group, thatcan be bonded via a linear or branched unsubstituted or at leastmonosubstituted C₁₋₅ alkylene group that can comprise at least oneheteroatom as a link and may be condensed with a five-membered orsix-membered monocyclic ring system; an unsubstituted or at leastmonosubstituted C₃₋₈-cycloaliphatic group that can comprise at least oneheteroatom as a ring member or that can be bonded via a linear orbranched unsubstituted or at least monosubstituted C₁₋₅ alkylene groupthat can comprise at least one heteroatom as a link and that can bebridged by a linear or branched unsubstituted or at leastmonosubstituted C.sub.1-5 alkylene group;

R⁵ represents a linear or branched unsubstituted or at leastmonosubstituted C₁₋₁₀ alkyl group that can comprise at least oneheteroatom as a link; a linear or branched unsubstituted or at leastmonosubstituted C₂₋₁₀ alkenyl group that can comprise at least oneheteroatom as a link; a linear or branched unsubstituted or at leastmonosubstituted C₂₋₁₀ alkynyl group that can comprise at least oneheteroatom as a link; an unsubstituted or at least monosubstitutedfive-membered to fourteen-membered aryl or heteroaryl group, that can bebonded via a linear or branched unsubstituted or at leastmonosubstituted C₁₋₅ alkylene group that can comprise at least oneheteroatom as a link; an unsubstituted or at least monosubstitutedC₃₋₈-cycloaliphatic group that can comprise at least one heteroatom as aring member and that can be bonded via a linear or branchedunsubstituted or at least monosubstituted C₁₋₅ alkylene group that cancomprise at least one heteroatom as a link; a —C(═O)OR⁸ group or a—C(═O)OR⁹ group either of that can be bonded via a linear or branchedC₁₋₁₀ alkylene group;

R⁶ represents an unsubstituted or at least monosubstituted five-memberedto fourteen-membered aryl or heteroaryl group, which aryl or heteroarylgroup may be bonded via a linear or branched unsubstituted or at leastmonosubstituted C₁₋₅ alkylene group that can comprise at least oneheteroatom as a link; an unsubstituted or at least monosubstitutedC₃₋₈-cycloaliphatic group that can comprise at least one heteroatom as aring member, or that can be bonded via a linear or branchedunsubstituted or at least monosubstituted C₁₋₅ alkylene group that cancomprise at least one heteroatom as a link; and

R⁷, R⁸, R⁹, R¹⁰, and R¹¹, independently represent a linear or branchedC₁₋₅ alkyl group, a linear or branched C₂₋₅ alkenyl group, or a linearor branched C₂₋₅ alkynyl group.

Compounds A, B and C whose structural formulas are shown below areillustrative preferred compounds of Series D.

A substituted 1-oxa-3,8-diazaspiro[4.5]-decan-2-one compound of Series Ecorresponds in structure to the formula

wherein

n is 1, 2, 3, 4 or 5;

R¹ denotes:

an optionally substituted 6- or 10-membered aryl group or an optionallysubstituted 5- to 14-membered heteroaryl group, wherein the aryl orheteroaryl group optionally can be fused with a saturated orunsaturated, optionally substituted mono- or bicyclic ring system;

R² denotes:

—C(═S)—NH—R³; —C(═O)—NH—R⁴; —S(═O)₂—R⁵; —(CH₂)—C(═O)—NH—R⁶;

—(CH₂)-D_(aa)-(CH₂)_(bb)-E_(cc)-(CH₂)_(dd)—R⁷, wherein

aa=0 or 1;

bb=0, 1 or 2;

cc=0 or 1; dd=0 or 1; and

the sum of aa and cc does not equal 0; and

D and E each independently denote O, S, NH, N(CH₃), N(C₂H₅) orN[CH(CH₃)₂];

—C(═O)—R⁸, or —S(═O)₂—NR¹⁹R¹⁰;

R³ denotes:

—(CHR¹¹)—(CH₂)_(w)—C(═O)—O—R¹², wherein w=0 or 1;

—(CHR¹³)—(CH₂)_(a)—K_(b)—(CH₂)_(c)-L_(d)-R¹⁴, wherein, a=0, 1 or 2; b=0or 1; c=0, 1 or 2; d=0 or 1, and K and L each independently denote O, S,NH, N(CH₃), N(C₂H₅) or N[CH(CH₃)₂];

a linear or branched, saturated or unsaturated, optionally substitutedC₁₋₁₀ aliphatic group;

an unsaturated or saturated, optionally substituted 3-, 4-, 5-, 6-, 7-,8- or 9-membered cycloaliphatic group which optionally can be bridgedwith 1 or 2 linear or branched, optionally substituted C₁₋₅ alkylenegroups or fused with a saturated, unsaturated or aromatic, optionallysubstituted mono- or bicyclic ring system, or both; or an optionallysubstituted 6- or 10-membered aryl group; or an optionally substituted5- to 14-membered heteroaryl group, wherein the aryl or heteroaryl groupoptionally can be fused with a saturated or unsaturated, optionallysubstituted mono- or bicyclic ring system;

R⁴ denotes:

—(CHR¹⁵)—(CH₂)_(e)-M_(f)-(CH₂)_(g)—P_(h)—R¹⁶, wherein e=0, 1 or 2; f=0or 1; g=0, 1 or 2; h=0 or 1; and M and P each independently denote O, S,NH, N(CH₃), N(C₂H₅) or N[CH(CH₃)₂];

a linear or branched, saturated or unsaturated, optionally substitutedC₁₋₁₀ aliphatic group;

an unsaturated or saturated, optionally substituted 3-, 4-, 5-, 6-, 7-,8- or 9-membered cycloaliphatic group which optionally can be bridgedwith 1 or 2 linear or branched, optionally substituted C₁₋₅ alkylenegroups or fused with a saturated, unsaturated or aromatic, optionallysubstituted mono- or bicyclic ring system, or both;

or an optionally substituted 6- or 10-membered aryl group or optionallysubstituted 5- to 14-membered heteroaryl group, wherein said aryl orheteroaryl group optionally can be fused with a saturated orunsaturated, optionally substituted mono- or bicyclic ring system;

R⁵ denotes:

—(CHR¹⁷)—(CH₂)_(k)-Q₁-(CH₂)_(m)-T_(o)-R¹⁸, wherein k=0, 1 or 2; 1=0 or1; m=0, 1 or 2; o=0 or 1; and Q and T each independently denote O, S,NH, N(CH₃), N(C₂H₅) or N[CH(CH₃)₂];

a linear or branched, saturated or unsaturated, optionally substitutedC₁₋₁₀ aliphatic group;

an unsaturated or saturated, optionally substituted 3-, 4-, 5-, 6-, 7-,8- or 9-membered cycloaliphatic group which optionally can be bridgedwith 1 or 2 linear or branched, optionally substituted C₁₋₅ alkylenegroups or fused with a saturated, unsaturated or aromatic, optionallysubstituted mono- or bicyclic ring system, or both; or an optionallysubstituted 6- or 10-membered aryl group or optionally substituted 5- to14-membered heteroaryl group, wherein the aryl or heteroaryl groupoptionally can be fused with a saturated or unsaturated, optionallysubstituted mono- or bicyclic ring system;

R⁶ denotes:

a linear or branched, saturated or unsaturated, optionally substitutedC₁₋₁₀ aliphatic group;

an unsaturated or saturated, optionally substituted 3-, 4-, 5-, 6-, 7-,8- or 9-membered cycloaliphatic group which optionally can be bridgedwith 1 or 2 linear or branched, optionally substituted C₁₋₅ alkylenegroups or fused with a saturated, unsaturated or aromatic, optionallysubstituted mono- or bicyclic ring system, or both; an optionallysubstituted 6- or 10-membered aryl group or optionally substituted 5- to14-membered heteroaryl group, wherein the aryl or heteroaryl groupoptionally can be fused with a saturated or unsaturated, optionallysubstituted mono- or bicyclic ring system;

R⁷ denotes:

a group selected from the group consisting of cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl,cycloheptenyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl,pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, thiomorpholinyl,tetrahydropyranyl, azepanyl, diazepanyl and dithiolanyl, wherein saidgroup optionally can be substituted with 1, 2, 3, 4 or 5 substituentsindependently selected from the group consisting of oxo (═O), thioxo(═S), —OH, —O—CH₃, —O—C₂H₅, —O—CH(CH₃)₂, —O—CH₂—CH₂—CH₃, —O—C(CH₃)₃,—O—CH₂—CH₂—CH₂—CH₃, —O—CF₃, —S—CF₃, —S—CF₂H, —S—CFH₂, methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,n-hexyl, n-heptyl, —C(═O)—CH₃, —C(═O)—C₂H₅, —C(═O)—C(CH₃)₃, —C(═O)—CF₃,—C(═O)—C₂F₅, —C(═O)—NH₂, —C(═O)—NH—CH₃, —C(═O)—NH—C₂H₅,—C(═O)—NH—C(CH₃)₃, —C(═O)—N(CH₃)₂, —C(═O)—N(C₂H₅)₂, —S(═O)₃—CH₃,—S(═O)₂—C₂H₅, —NH—S(═O)₂—CH₃, —S(˜0)₂—NH—CH₃ and —S(═O)₂—NH₂;

or a group selected from the group consisting of phenyl, naphthyl, and[1,2,3,4]-tetrahydronaphthyl, wherein said group optionally can besubstituted with 1, 2, 3, 4 or 5 substituents independently selectedfrom the group consisting of F, Cl, Br, I, —CN, —CF₃, —SF₅, —OH, —O—CH₃,—O—C₂H₅, —O—CH(CH₃)₃, —O—CH₂—CH₂—CH₃, —O—C(CH₃)₃, —O—CH₂—CH₂—CH₂—CH₃,—NO₂, —O—CF₃, —S—CF₃, —S—CF₂H, —S—CFH₂, SH, —S—CH₃, —S—C₂H₅,—S—CH(CH₃)₂, —S—CH₂—CH₂—CH₃, —S—C(CH₃)₃, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,—C(CH₃)₂—C₂H₅, n-hexyl, n-heptyl, —NH—C(═O)—O—CH₃, —NH—C(═O)—O—C₂H₅,—NH—C(═O)—O—C(CH₃)₃, —O—CH₂—CH₃—CH₂—CH₃, —NH—C(═O)—CH₃, —NH—C(═O)—C₂H₅,—NH—C(═O)—C(CH₃)₃, —C(═O)—OH, —(CH₂)—C(═O)—OH, —C(═O)—O—CH₃,—C(═O)—O—CH₂—CH₃, —C(═O)—O—CH(CH₃)₂, —C(═O)—O—C(CH₃)₃, —NH—CH₃,—NH—C₂H₅, —NH—CH(CH₃)₂, —NH—C(CH₃)₃, —N(CH₃)₂, —N(C₂H₅)₂, —N(CH₃)(C₂H₅),—C(═O)—H, —C(═O)—CH₃, —C(═O)—C₂H₅, —C(═O)—C(CH₃)₃, —C(═O)—CF₃,—C(═O)—C₂F₅, —C(═O)—NH₃, —C(═O)—NH—CH₃, —C(═O)—NH—C₂H₅,—C(═O)—NH—C(CH₃)₃, —C(═O)—N(CH₃)₂, —C(═O)—N(C₂H₅)₂, —S(═O)₂—CH₃,—S(═O)₂—C₂H₅, —NH—S(═O)₂—CH₃, —S(═O)₃—NH—CH₃, —S(═O)₂—NH₂,—S(═O)₂—NH-phenyl, phenyl and benzyl, wherein the cyclic moiety of thegroups —S(═O)₂—NH-phenyl, phenyl and benzyl optionally can besubstituted with 1, 2, 3, 4 or 5 substituents independently selectedfrom the group consisting of F, Cl, Br, —OH, —CF₃, —SF₅, —NO₂, methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, n-hexyl, n-heptyl, —O—CH₃, —O—C₂H₅, —O—CH(CH₃)₃,—O—CH₂—CH₃—CH₃, —O—C(CH₃)₃, —O—CH₂—CH₂—CH₂—CH₃, —O—CF₃, —S—CF₃, phenyland —O-benzyl;

R⁸ denotes

—(CHR¹⁹)—V_(p)—(CH₂)_(q)—(CH₂)_(r)—W_(s)—R²⁰, wherein

p=0 or 1;

g=0, 1 or 2;

r=0, 1 or 2;

s=0 or 1; and

V and W each independently denote O, S, NH, —NH—CH₃, —NH—C₂H₅,—NH—CH(CH₃)₂;

—(CH═CH)—R²¹;

—(CR²²R²³)—Y_(t)—(CR²⁴R²⁵)_(u)—(CH₂)_(v)—C(═O)—OR²⁶, wherein

t=0 or 1, u=0 or 1;

v=0 or 1, and Y denotes O, S, NH, —NH—CH₃, —NH—C₂H₅, —NH—CH(CH₃)₂;

—(CHR²⁷)—O—C(═O)—R²⁸;

—CH[(CH₂)R²⁹][NH—S(═O)₂—R³⁰];

—CH[(CH₂)R³¹][NH—C(═O)—O—R³²];

a linear or branched, saturated or unsaturated, optionally substitutedC₁₋₁₀ aliphatic group selected from the group consisting of methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, sec-pentyl, 3-pentyl, —(CH₂)—(CH₂)—(C(CH₃)₃), n-hexyl,2-hexyl, 3-hexyl, n-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, n-octyl,—(CH₂)—(CH)(C₂H₅)—(CH₂)—(CH₂)—(CH₂)—(CH₃), vinyl, 1-propenyl,2-propenyl, 1-butenyl, 2-butenyl and 3-butenyl;

an unsaturated or saturated, optionally substituted 3-, 4-, 5-, 6-, 7-,8- or 9-membered cycloaliphatic group that optionally can be bridgedwith 1 or 2 linear or branched, optionally substituted C₁₋₅ alkylenegroups, or fused with a saturated, unsaturated or aromatic, optionallysubstituted mono- or bicyclic ring system, or both; or an optionallysubstituted 6- or 10-membered aryl group or optionally substituted 5- to14-membered heteroaryl group, wherein the aryl or heteroaryl groupoptionally can be fused with a saturated or unsaturated, optionallysubstituted mono- or bicyclic ring system;

R⁹ and R¹⁰ each independently denote a linear or branched, saturated orunsaturated, optionally substituted C₁₋₁₀ aliphatic group;

R¹¹, R¹³, R¹⁵, R¹⁷, R¹⁹, R²², R²³, R²⁴, R²⁵ and R²⁶ each independentlydenote a hydrogen or a linear or branched, saturated or unsaturated,optionally substituted C₁₋₁₀ aliphatic group;

R¹², R²⁸ and R³² each independently denote a linear or branched,saturated or unsaturated, optionally substituted C₁₋₁₀ aliphatic group;

R¹⁴, R¹⁶, R¹⁸ and R²⁰ each independently denote a linear or branched,saturated or unsaturated, optionally substituted C₁₋₁₀ aliphatic group;

an unsaturated or saturated, optionally substituted 3-, 4-, 5-, 6-, 7-,8- or 9-membered cycloaliphatic group that optionally can be bridgedwith 1 or 2 linear or branched, optionally substituted C₁₋₅ alkylenegroups, or fused with a saturated, unsaturated or aromatic, optionallysubstituted mono- or bicyclic ring system, or both; or

an optionally substituted 6- or 10-membered aryl group or optionallysubstituted 5- to 14-membered heteroaryl group, wherein the aryl orheteroaryl group optionally can be fused with a saturated orunsaturated, optionally substituted mono- or bicyclic ring system; and

R²¹, R²⁷, R²⁹, R³⁰ and R³¹ each independently denote an unsaturated orsaturated, optionally substituted 3-, 4-, 5-, 6-, 7-, 8- or 9-memberedcycloaliphatic group which optionally can be bridged with 1 or 2 linearor branched, optionally substituted C₁₋₅ alkylene groups, or fused witha saturated, unsaturated or aromatic, optionally substituted mono- orbicyclic ring system, or both; or an optionally substituted 6- or10-membered aryl group or optionally substituted 5- to 14-memberedheteroaryl group, wherein the aryl or heteroaryl group optionally can befused with a saturated or unsaturated, optionally substituted mono- orbicyclic ring system; wherein the above-stated C₁₋₁₀ aliphatic groupseach independently may optionally be substituted with 1, 2, 3, 4 or 5substituents independently selected from the group consisting of F, Cl,Br, I, —CN, —NO₂, —OH, —SH and —NH₂;

the above-stated cycloaliphatic groups each independently can optionallybe substituted with 1, 2, 3, 4 or 5 substituents independently selectedfrom the group consisting of oxo (═O), thioxo (═S), F, Cl, Br, I, —CN,—CF₃, —SF₅, —OH, —O—C₁₋₅alkyl, —NH₂, —NO₂, —O—CF₃, —S—CF₃, —S—CF₂H,—S—CFH₂, —SH, —S—C₁₋₅-alkyl, —C₁₋₅alkyl, —C(═O)—OH, —(CH₂)—C(═O)—OH,—C(═O)—O—C₁₋₅alkyl, —(CH₂)—C(═O)—O—C₁₋₅alkyl, —O—C(═O)—C₁₋₅alkyl,—NH—C₁₋₅alkyl, —N(C₁₋₅alkyl)₂, —NH-phenyl, —NH-pyridinyl,—N(C₁₋₅alkyl)-phenyl, —N(C₁₋₅alkyl)-pyridinyl, —NH—C(═O)—O—C₁₋₅alkyl,—C(═O)—H, —C(═O)—C₁₋₅alkyl, —C(═O)—C₁₋₅-perfluoroalkyl, —C(═O)—NH₂,—C(═O)—NH—C₁₋₅alkyl, C(═O)—N-(C₁₋₅alkyl)₂, —S(═O)₂—C₁₋₅alkyl,—S(═O)₂-phenyl, —NH—S(═O)₂—C₁₋₅alkyl, —S(═O)₂—NH—C₁₋₅alkyl, —S(═O)₂—NH₂,—S(═O)₂—NH-phenyl, cyclohexyl, cyclopentyl, pyridinyl,[1,2,5]-thiadiazolyl, pyridazinyl, —(CH₂)— benzo[b]furanyl, —O-phenyl,—O-benzyl, phenyl and benzyl, wherein the cyclic moiety of the groups—S(═O)₂—NH-phenyl, —NH-phenyl, —NH-pyridinyl, —N(C₁₋₅alkyl)phenyl,—N(C₁₋₅alkyl)pyridinyl, pyridinyl, cyclopentyl, [1,2,5]-thiadiazolyl,cyclohexyl, pyridazinyl, —S(═O)₂-phenyl, —O-phenyl, —O-benzyl, phenyl,—(CH₂)-benzo[b]furanyl and benzyl optionally can be substituted with 1,2, 3, 4 or 5 substituents independently selected from the groupconsisting of F, Cl, Br, —OH, —CF₃, —SF₅, —CN, —NO₂, —C₁₋₅alkyl,—O—C₁₋₅alkyl, —O—CF₃, —S—CF₃, phenyl and —O-benzyl, and comprise 1, 2,3, 4 or 5 heteroatom(s) mutually independently selected from the groupconsisting of oxygen, nitrogen and sulfur;

the above-stated C₁₋₅alkylene groups each independently may optionallybe substituted with 1, 2, 3, 4 or 5 substituents independently selectedfrom the group consisting of F, Cl, Br, I, —CN, —NO₂, —OH, —SH and —NH₂;

the rings of the above-stated mono- or polycyclic ring systems eachindependently may optionally be substituted with 1, 2, 3, 4 or 5substituents independently selected from the group consisting of oxo(═O), thioxo (═S), F, Cl, Br, I, —CN, —CF₃, —SF₅, —OH, —O—C₁₋₅alkyl-NH₂,—NO₂, —O—CF₃, —S—CF₃, —S—CF₂H, —S—CFH₂, —SH, —S—C₁₋₅alkyl, —C₁₋₅alkyl,—C(═O)—OH, —(CH₂)—C(═O)—OH, C₁₋₅alkyl, —(CH₂)—C(═O)—O—C₁₋₅alkyl,—O—C(═O)—C₁₋₅alkyl, —NH—C₁₋₅alkyl, —N(C₁₋₅alkyl)₂, —NH-phenyl,—NH-pyridinyl, —N(C₁₋₅alkyl)-phenyl, —N(C₁₋₅alkyl)-pyridinyl,—NH—C(═O)—O—C₁₋₅alkyl, —C(═O)—H, —C(═O)—C₁₋₅alkyl,—C(═O)—C₁₋₅-perfluoroalkyl, —C(═O)—NH₂, —C(═O)—NH—C₁₋₅alkyl,C(═O)—N—(C₁₋₅alkyl)₂, —S(═O)₂—C₁₋₅alkyl, —S(═O)₂-phenyl,—NH—S(═O)₂—C₁₋₅alkyl, —S(═O)₂—NH—C₁₋₅alkyl, —S(═O)₂—NH₂,—S(═O)₂—NH-phenyl, cyclohexyl, cyclopentyl, pyridinyl,[1,2,5]-thiadiazolyl, pyridazinyl, —(CH₂)-benzo[b]furanyl, —O-phenyl,—O-benzyl, phenyl and benzyl,

wherein the cyclic moiety of the groups —S(═O)₂—NH-phenyl, —NH-phenyl,—NH-pyridinyl, —N(C₁₋₅alkyl)phenyl, —N(C₁₋₅alkyl)pyridinyl, pyridinyl,cyclopentyl, [1,2,5]-thiadiazolyl, cyclohexyl, pyridazinyl,—S(═O)₂-phenyl, —O-phenyl, —O-benzyl, phenyl, —(CH₂)-benzo[b]furanyl andbenzyl optionally can be substituted with 1, 2, 3, 4 or 5 substituentsindependently selected from the group consisting of F, Cl, Br, —OH,—CF₃, —SF₅, —CN, —NO₂, C₁₋₅alkyl, —O—C₁₋₅alkyl, —NH₂, —O—CF₃, —S—CF₃,phenyl and —O-benzyl;

the rings of the above-stated mono- or bicyclic ring systems are eachindependently 5-, 6- or 7-membered and each independently may optionallycomprise as ring member(s), 1, 2, 3, 4 or 5 heteroatom(s) independentlyselected from the group consisting of oxygen, nitrogen and sulfur; theabove-stated aryl or heteroaryl groups each independently may optionallybe substituted with 1, 2, 3, 4 or 5 substituents independently selectedfrom the group consisting of F, Cl, Br, I, —CN, —CF₃, —SF₅, —OH,—O—C₁₋₅alkyl-NH₂, —NO₂, —O—CF₃, —S—CF₃, —S—CF₂H, —S—CFH₂, —SH,—S—C₁₋₅-alkyl, —C₁₋₅alkyl, —C(═O)—OH, —(CH₂)—C(═O)—OH,—C(═O)—O—C₁₋₅alkyl, —(CH₂)—C(═O)-β—C₁₋₅alkyl, —O—C(═O)—C₁₋₅alkyl,—NH—C₁₋₅alkyl, —N(C₁₋₅alkyl)₂, —NH—C(═O)—O—C₁₋₅alkyl,—NH—C(═O)—C₁₋₅alkyl, —C(═O)—H, —C(═O)—C₁₋₅-alkyl,—C(═O)—C₁₋₅-perfluoroalkyl, —C(═O)—NH₂, —C(═O)—NH—C₁₋₅alkyl,C(═O)—N—(C₁₋₅alkyl)₂, —S(═O)₂—C₁₋₅alkyl, —S(═O)₂-phenyl,—NH—S(═O)₂—C₁₋₅alkyl, —S(═O)₂—NH—C₁₋₅alkyl, —S(═O)₂—NH₂,—S(═O)₂—NH-phenyl, cyclohexyl, cyclopentyl, pyridinyl,—(CH₂)-benzo[b]furanyl, —O-phenyl, —O-benzyl, phenyl and benzyl, whereinthe cyclic moiety of the groups pyridinyl, cyclopentyl, cyclohexyl,pyridazinyl, —S(═O)₂-phenyl, —S(═O)₂—NH-phenyl, —O-phenyl, —O-benzyl,phenyl, —(CH₂)— benzo[b]furanyl, optionally can be substituted with 1,2, 3, 4 or 5 substituents independently selected from the groupconsisting of F, Cl, Br, —OH, —CF₃, —SF₅, —NO₂, C₁₋₅alkyl, —O—C₁₋₅alkyl,—O—CF₃, —S—CF₃, phenyl and -o-benzyl;

and the above-stated heteroaryl groups each independently may optionallycomprise as ring member(s), 1, 2, 3, 4 or 5 heteroatom(s) independentlyselected from the group consisting of oxygen, nitrogen and sulfur; or apharmaceutically acceptable salt or solvate thereof.

Preferably, in a contemplated compound, R¹ denotes a group selected fromthe group consisting of phenyl, naphthyl, (1,3)-benzodioxolyl,(1,4)-benzodioxanyl, 2H-chromenyl, thiophenyl, furanyl, pyrrolyl,pyrazolyl, pyrazinyl, pyranyl, triazolyl, pyridinyl, imidazolyl,indolyl, isoindolyl, benzo[b]furanyl, benzo[b]thiophenyl, thiazolyl,[1,2,3]-thiadiazolyl, [1,2,4]-oxadiazolyl, benzo[2,1,3]thiadiazolyl,[1,2,3]-benzothiadiazolyl, [2,1,3]-benzoxadiazolyl,[1,2,3]-benzoxadiazolyl, [1,2,3,4]-tetrahydronaphthyl,[1,2,3,4]-tetra-hydroquinolinyl, [1,2,3,4]-tetrahydroisoquinolinyl,[1,2,3,4]-tetrahydroquinazolinyl, [3,4]-dihydro-2H-1,4-benzoxazinyl,oxazolyl, isoxazolyl, pyridazinyl, pyrazinyl, pyrimidinyl, indazolyl,quinazolinyl, quinolinyl and isoquinolinyl. That R¹ group can optionallybe substituted with 1, 2, 3, 4 or 5 substituents independently selectedfrom the group consisting of F, 01, Br, I, —CN, —CF₃, —SF₅, —OH, —O—CH₃,—O—C₂H₅, —O—CH(CH₃)₂, —O—CH₂—CH₂—CH₃, —O—C(CH₃)₃, —O—CH₂—CH₂—CH₂—CH₃,—NO₂, —O—CF₃, —S—CF₃, —S—CF₂H, —S—CFH₂, —SH, —S—CH₃, —S—C₂H₅,—S—CH(CH₃)₂, —S—CH₂—CH₂—CH₃, —S—C(CH₃)₃, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl,n-heptyl, —NH—C(═O)—O—CH₃, —NH—C(═O)—O—C₂H₅, —NH—C(═O)—O—C(CH₃)₃,—NH—C(═O)—CH₃, —NH—C(═O)—C₂H₅, —NH—C(═O)—C(CH₃)₃, —C(═O)—H, —C(═O)—CH₃,—C(═O)—C₂H₅, —C(═O)—C(CH₃)₃, —C(═O)—CF₃, —C(═O)—C₂F₅, —C(═O)—NH₂,—C(═O)—NH—CH₃, —C(═O)—NH—C₂H₅, —C(═O)—NH—C(CH₃)₃, —C(═O)—N(CH₃)₂,—C(═O)—N(C₂H₅)₂, —S(═O)₃—CH₃, —S(═O)₃—C₂H₅, —NH—S(═O)₂—CH₃,—S(═O)₃—NH—CH₃, —S(═O)₂—NH₂, —S(═O)₂—NH-phenyl and -benzyl, wherein thecyclic moiety of each phenyl or benzyl group independently canoptionally be substituted with 1,2,3,4, or 5 substituents independentlyselected from the group consisting of F, Cl, Br, —CF₃, —SF₅, —NO₂,methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, n-pentyl, n-hexyl, n-heptyl, —O—CH₃, —O—C₂H₅, —O—CH(CH₃)₃,—O—CH₂—CH₂—CH₃, —O—C(CH₃)₃, —O—CH₂—CH₂—CH₂—CH₃, —O—CF₃, —S—CF₃, phenyland —O-benzyl.

It is also preferred that the R² group of a contemplated compound denoteC(═S)—NH—R³; —C(═O)—NH—R⁴; —S(═O)₂R⁵; —(CH₂)—C(═O)—NH—R⁶; —(CH₂)—O—R⁷,—(CH₂)—S—R⁷, —(CH₂)—NH—R⁷, —(CH₂)—N(CH₃)—R⁷, —(CH₂)—(CH₂)—O—R⁷,—(CH₂)—(CH₂)—S—R⁷, —(CH₂)—NH—R⁷, —(CH₂)—N(CH₃)—R⁷,—(CH₂)—(CH₂)—(CH₂)—O—R⁷, —(CH₂)—(CH₂)—(CH₂)—S—R⁷,—(CH₂)—(CH₂)—(CH₂)—NH—R⁷, —(CH₂)—(CH₂)—(CH₂)—N(CH₃)—R⁷,—(CH₂)—O—(CH₂)—R⁷, —(CH₂)—S—(CH₂)—R⁷, —(CH₂)—NH—(CH₂)—R⁷, —C(═O)—R⁸, or—S(═O)₂—NR⁹R¹⁰.

Pharmaceutical Compositions

A contemplated compound useful in the invention can be provided for useby itself, or as a pharmaceutically acceptable salt. Regardless ofwhether in the form of a salt or not, a contemplated composition istypically dissolved or dispersed in a pharmaceutically acceptablediluent that forms a pharmaceutical composition and that pharmaceuticalcomposition is administered the CNS and/or other cells.

A contemplated compound can be used in the manufacture of a medicament(pharmaceutical composition) that is useful at least for inhibiting tauprotein phosphorylation in mammalian cells and mammalian cellpreparations. A contemplated compound can be used in the manufacture ofa medicament that is useful at least for inhibiting the interaction ofFLNA with α7nAChR and TLR4, as well as of Aβ₄₂ with α7nAChR in mammaliancells and mammalian cell preparations.

A contemplated pharmaceutical composition contains an effective amountof a contemplated compound or a pharmaceutically acceptable salt thereofdissolved or dispersed in a physiologically tolerable carrier. Such acomposition can be administered to mammalian cells in vitro as in a cellculture, or in vivo as in a living, host mammal in need.

A contemplated composition is typically administered a plurality oftimes over a period of days. More usually, a contemplated composition isadministered once or twice daily. It is contemplated that onceadministration of a contemplated compound has begun the compound will beadministered chronically for the duration of the study being carried outor for a recipient's lifetime.

A contemplated compound can bind to FLNA at a 100 femtomolarconcentration and effectively inhibit cytokine release fromLPS-stimulated astrocytes in vitro. A contemplated compound is moreusually utilized at picomolar to micromolar amounts. Thus, an effectiveamount of a contemplated compound present in a contemplatedpharmaceutical composition is that which provides a concentration ofabout 100 femtomolar to about micromolar to a host animal's blood streamor to an in vitro cell medium in practicing a contemplated method of theinvention. A more usual amount is about picomolar to about micromolar. Astill more usual amount is about picomolar to about nanomolar. A skilledworker can readily determine an appropriate dosage level of acontemplated compound to inhibit a desired amount of tau proteinphosphorylation.

A contemplated pharmaceutical composition can be administered orally(perorally), parenterally, by inhalation spray in a formulationcontaining conventional nontoxic pharmaceutically acceptable carriers,adjuvants, and vehicles as desired. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intrasternal injection, or infusion techniques. Formulation of drugs isdiscussed in, for example, Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa.; 1975 and Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980.

For injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that can be employed are water, Ringer's solution,and isotonic sodium chloride solution, phosphate-buffered saline. Liquidpharmaceutical compositions include, for example, solutions suitable forparenteral administration. Sterile water solutions of an activecomponent or sterile solution of the active component in solventscomprising water, ethanol, or propylene glycol are examples of liquidcompositions suitable for parenteral administration.

In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables. Dimethyl acetamide, surfactants including ionic andnon-ionic detergents, polyethylene glycols can be used. Mixtures ofsolvents and wetting agents such as those discussed above are alsouseful.

Sterile solutions can be prepared by dissolving the active component inthe desired solvent system, and then passing the resulting solutionthrough a membrane filter to sterilize it or, alternatively, bydissolving the sterile compound in a previously sterilized solvent understerile conditions.

Solid dosage forms for oral administration can include capsules,tablets, pills, powders, and granules. In such solid dosage forms, acontemplated compound is ordinarily combined with one or more excipientsappropriate to the indicated route of administration. If administeredper os, the compounds can be admixed with lactose, sucrose, starchpowder, cellulose esters of alkanoic acids, cellulose alkyl esters,talc, stearic acid, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets can contain a controlled-release formulation as canbe provided in a dispersion of active compound in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering agents such as sodium citrate, magnesium orcalcium carbonate or bicarbonate. Tablets, capsules and pills canadditionally be prepared with enteric coatings.

A mammal in need of treatment and to which a pharmaceutical compositioncontaining a contemplated compound is administered can be a primate suchas a human, an ape such as a chimpanzee or gorilla, a monkey such as acynomolgus monkey or a macaque, a laboratory animal such as a rat, mouseor rabbit, a companion animal such as a dog, cat, horse, or a foodanimal such as a cow or steer, sheep, lamb, pig, goat, llama or thelike. Where in vitro mammalian cell contact is contemplated, a CNStissue culture of cells from an illustrative mammal is often utilized,as is illustrated hereinafter.

Preferably, the pharmaceutical composition is in unit dosage form. Insuch form, the composition is divided into unit doses containingappropriate quantities of the active agent. The unit dosage form can bea packaged preparation, the package containing discrete quantities ofthe preparation, for example, in vials or ampules.

Several useful contemplated compounds are amines and can typically beused in the form of a pharmaceutically acceptable acid addition saltderived from an inorganic or organic acid. Exemplary salts include butare not limited to the following: acetate, adipate, alginate, citrate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate,ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxy-ethanesulfonate, lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, mesylate andundecanoate.

Other compounds useful in this invention that contain acidfunctionalities can also form salts with a base. Illustrative basesinclude amine bases such as mono-, di- and tri-C₁-C₄-alkyl orhydroxyalkyl amines like triethyl amine, dimethylamine,2-hydroxyethylamine, and dimethyl-2-hydroxyethylamine, and bases such asalkali metal, alkaline earth metal quaternary C₁-C₆-alkyl ammoniumhydroxides, such as sodium, potassium, calcium, magnesium andtetramethylammonium hydroxides. Basic salts such as alkali metal oralkaline earth metal and ammonium carbonates and phosphates are alsocontemplated.

The reader is directed to Berge, J. Pharm. Sci. 68(1):1-19 (1977) forlists of commonly used pharmaceutically acceptable acids and bases thatform pharmaceutically acceptable salts with pharmaceutical compounds.

In some cases, the salts can also be used as an aid in the isolation,purification or resolution of the compounds of this invention. In suchuses, the acid used and the salt prepared need not be pharmaceuticallyacceptable.

Discussion

The discussion that follows illustrates compounds and compositions thatcontain one or more of those compounds that bind the scaffolding proteinFLNA, and particularly the VAKGL (SEQ ID NO: 1) pentapeptide bindingsite present in the FLNA protein, and inhibit the phosphorylation of thetau protein. Some compounds of such compositions also disrupt the toxicsignaling of amyloid-β₄₂ (Aβ₄₂), as well as reduce the inflammationcaused by both Aβ₄₂ and ongoing neurodegeneration. These compoundsdiminish many aspects of AD-like pathology, including impairments innormal receptor functioning.

Initial studies were carried out with compounds of each of the fourstructural series using Compounds A0033, A0040, A0053, A0068, B0055,C0105, C0114, C0137 and C0138 as illustrative or exemplary. Additionalstudies were also carried out using Compounds C0134, Compound A,Compound B, and Compound C. The results shown in the Figures anddiscussed hereinafter are indicative of the generality of resultsobtained using these structurally very different compounds. Initialresults indicated that the compounds appear to be orally available andwell tolerated because notable plasma and CNS levels were produced butnegligible side effects were noted at 2 g/kg administered orally inrats. Those results also indicated similar activities among the ninecompounds, with Compounds C0105 and C0114 being used for further studiesbecause of their high activity, ease of synthesis, solubility andabsence of enantiomers.

The fact that Aβ₄₂ binding blocks Ca⁺² influx by α7nAChRs [Wang et al.,J Neurosci 35:10961-10973 (2009); Wang et al., Biol Psychiatry67:522-530 (2010)] suggests that one conformational change in α7nAChRsmay occur in the interface between extracellular and transmembranedomains, the area governing channel opening/desensitization [Bouzat etal., J Neurosci 28:7808-7819 (2008)]. This conformational change likelyexposes a positive charge-rich transmembrane region close to the Aβ₄₂binding site. FLNA binds this positive charge to stabilize the boundAβ₄₂ and additional binding of Aβ₄₂ peptides, leading to eventualinternalization of Aβ₄₂-α7nAChR complexes [(Nagele et al., Neuroscience110:199-211 (2002)]. Compound C0105 disruption of the FLNA-α7nAChRinteraction stops the pathological signaling and stops Aβ₄₂high-affinity anchoring to the receptor.

Using organotypic frontocortical slice cultures of adult rats, Aβsignaling through the α7 nicotinic acetylcholine receptor (α7nAChR) isshown to require the recruitment of FLNA. By binding a criticalpentapeptide segment of FLNA, these compounds block the FLNA-α7nAChRassociation and the signaling cascade of Aβ₄₂. In the illustrativeAβ₄₂-treated organotypic frontocortical slice cultures, exemplaryCompounds C0105 and C0114 each separately dramatically reducephosphorylation of tau at all three phosphorylation sites of tau foundin neurofibrillary tangles (FIGS. 4D, 6B and 6C), and fully restorenormal functioning of α7nAChR and downstream N-methyl-D-aspartatereceptors (NMDARs), critical for learning and memory.

Further, indicative of the insulin resistance common to AD patients[Neumann et al., Curr Alzheimer Res 5:438-447 (2008)], the function ofAβ₄₂-impaired IRs in the illustrative slice cultures was also restoredby each of exemplary Compounds C0105 and C0114 (FIG. 5B). Thesecompounds, with a novel mechanism of disrupting the toxic signaling ofAβ₄₂, confer a restoration of multiple Aβ₄₂-induced dysfunctions.Moreover, disabling the Aβ-induced α7nAChR signaling without directlyaffecting α7nAChRs avoids altering the sensitivity or cell surface levelof the receptors, an insidious problem with using chronic receptoragonists or antagonists.

In an ICV Aβ₄₂ infusion mouse model of Alzheimer's disease, CompoundC0105 greatly reduced tau phosphorylation, multiple Aβ₄₂-inducedsignaling impairments, inflammatory cytokine release as well asneurofibrillary tangles and Aβ₄₂ aggregates. Illustrative Compound C0105accomplished all these effects via its high-affinity binding to FLNA.Aβ₂ dramatically increases FLNA association with α7nAChR to enable itsbinding and toxic signaling through this receptor, and illustrativeCompound C0105 prevented this cascade.

The anti-inflammatory effect of Compound C0105 occurs by a similardisruption of Aβ₄₂-induced FLNA association with TLR4. Aβ₄₂ increasesFLNA association with TLR4, and this association appears to be criticalto inflammatory cytokine production due to Aβ₄₂ exposure, becauseillustrative Compound C0105 nearly abolishes this cytokine production.Although Aβ₄₂ does not itself interact with TLR4, Aβ₄₂ binds to CD14,which in turn binds TLR4 to produce the inflammation noted in AD[Reed-Geaghan et al., J. Neurosci. 29(38):11982-11992 (Sep. 23, 2009)].

The normalization of function of α7nAChR, NMDAR and IRs furtherillustrates the broad spectrum of benefits of a contemplated compoundsuch as Compound C0105 as a potential therapeutic for AD. AlthoughNMDARs are downstream of α7nAChR and are likely compromised as a directresult of the Aβ₄₂ toxic signaling via α7nAChR, IRs are not directlydownstream of α7nAChR in neural networks. Additionally, although FLNAinteracts with IR, this interaction is unchanged by Aβ₄₂ or CompoundC0105. Nevertheless, Aβ₄₂ impairs IR signaling, and a contemplatedcompound such as Compound C0105 restores that signaling, suggesting atherapeutic effect on this component of AD as well.

Current thinking points to the need for several different simultaneousapproaches to treat AD. With its multitude of therapeutic effects andnovel target, a contemplated compound such as Compound C0105 has greatpotential as a disease-modifying therapeutic for AD.

The toxicities of Aβ₄₂ in AD as well as in the mild cognitive impairmentof senile dementia are believed by many in the field to be due to itscapacity to signal through α7nAChR. This toxic signaling activates ERK2and phosphorylates tau, a critical component of NFTs. The novelFLNA-binding compounds presented here potently suppress Aβ₄₂ signalingthrough the α7nAChR at nanomolar (nM) or sub-nM concentrations. Thesecompounds accomplish this blockade of signaling by preventing theincreased association of FLNA with α7nAChR caused by Aβ₄₂. Specifically,their binding may alter the conformation of FLNA so that it is notrecruited to the receptor.

Further, the disabling of this interaction appears also to decrease theaffinity of Aβ₄₂ binding to α7nAChR, as shown by decreased binding ofFITC-Aβ₄₂ in the presence of a contemplated compound such as compoundsC0105 or C0114. The resulting blockade of signaling was evidenced bydecreased ERK2 activation and decreased tau phosphorylation at all threephosphorylation sites of tau found in NFTs in AD brains.

In addition to disrupting the FLNA-α7nAChR association that is increasedby Aβ₄₂, illustrative Compounds C0105 and C0114 prevent an Aβ₄₂-inducedassociation of FLNA with TLR4 (FIG. 5), the immune receptor responsiblefor cytokine release. This association, along with the ongoingneurodegeneration, may drive the massive inflammation in AD brains, [Leeet al., Arch Pharm Res 33:1539-1556 (2010)] and its disruption is theprobable mechanism of action for anti-inflammatory effects of ourFLNA-binding compounds [Burns et al., Recent Patents on CNS DrugDiscovery 5:210-220 (2010)].

The anti-inflammatory activity of illustrative Compound C0105 wasdemonstrated in an ICV Aβ₄₂ infusion mouse model of AD. Levels of IL-6,TNF-α and IL-1β were decreased by 80-100% in mice receiving C0105. Inaddition, previous results showed that other compounds that bind thesame pentapeptide region of FLNA dramatically decreased inflammatorycytokine release from LPS-stimulated primary human astrocytes [Burns etal., Recent Patents on CNS Drug Discovery 5:210-220 (2010)]. Ananti-inflammatory property should be a great benefit in an ADtherapeutic.

The fact that a contemplated compound such as Compounds C0105 and C0114also restore normal functioning of the downstream NMDAR (FIG. 8) and theIR suggests that the benefits of preventing Aβ signaling via the α7nAChRare not isolated to the health of α7nAChRs, but instead can be thecritical point of pathogenesis in AD. Because NMDAR signaling isessential for long-term potentiation (LTP) and hence learning andmemory, maintaining normal functioning of this neurotransmitter systemis crucial for preserving memory in AD.

The reason that these compounds preserve IR function remains speculativebecause the FLNA-IR linkage is not affected by Aβ₄₂, nor by thesecompounds and because IRs are not directly downstream of α7nAChRs.Nevertheless, preserving sensitivity of IRs to insulin (FIG. 10)represents a divergent benefit of preventing the toxic signaling of Aβthrough α7nAChRs in AD pathology. Moreover, the FLNA-binding compoundspresented here represent a novel and perhaps safer approach topreventing this toxic signaling without directly antagonizing ordesensitizing the receptor.

Furthermore, their efficacy at low nM concentrations indicates, a largewindow for therapeutic efficacy. Additionally, the ability to removebound Aβ from α7nAChRs by decreasing the normally high affinity to thereceptor [Wang et al., J Biol Chem 275:5626-5632 (2000)] suggests that acontemplated compound can be effective not just in preventing AD but canalso provide some cognitive recovery and help stop further degenerationin later stages of AD.

Twice daily intraperitoneal administration to E129 mice with 10 mg/kg ofillustrative Compound C0105 greatly reduced Aβ₄₂-induced increases inFLNA associations with both α7nAChR and TLR4, suppressed tauphosphorylation at all three phosphorylation sites of tau found inneurofibrillary tangles, reduced the level of Aβ₄₂-α7nAChR complexes,prevented the Aβ₄₂-induced functional impairments in α7nAChR, NMDAR andIR signaling, and suppressed inflammatory cytokine levels (FIGS. 14-24).Illustrative Compound C0105 accomplished all these effects via itshigh-affinity binding to FLNA. Aβ₄₂ dramatically increases FLNAassociation with α7nAChR to enable its binding and toxic signalingthrough this receptor.

Specific Results

Increased α7nAChR-FLNA Coupling in Frontal Cortex of AD Transgenic Miceand AD Patients

Incubation of synaptosomes with Aβ₄₂ in vitro increases α7nAChR couplingto a scaffolding protein with 300 KDa molecular mass, which wassuspected to be FLNA. Although FLNA is known to couple with manyreceptor proteins, this data is the first to reveal the α7nAChR-FLNAconnection and suggests that AD, with elevated Aβ₄₂ burdens, may haveincreased α7nAChR-FLNA coupling. This hypothesis was directly tested insynaptosomes prepared from frontal cortices of 6-month-old AD transgenicand wild-type mice, as well as AD-control human pairs matched closelyfor the age and postmortem delays.

The data presented in FIG. 1 show a 3-fold increase in the abundance ofα7nAChR-FLNA complexes in frontal cortex of both AD transgenic mice andAD patients. Because FLNA is known to regulate signaling of itsassociated receptors, increases in the α7nAChR-FLNA association inducedby Aβ₄₂ can be related to Aβ₄₂-evoked α7nAChR signaling and tauphosphorylation. Thus, compounds that reduce Aβ₄₂-elicited α7nAChR-FLNAassociation may reduce neurofibrillary pathology in AD.

Ex Vivo in Synaptosomes

High-Affinity FLNA-Binding Compounds Reduce Aβ₄₂-Induced α7nAChR-FLNAAssociation, ERK2 Activation and Tau Phosphorylation Ex Vivo inSynaptosomes

Nine high-affinity FLNA-binding compounds [A0033, A0040, A0053, A0068,B0055, C0105, C0114, C0137 and 00138] were assayed to determine whetherthey could disrupt the association of FLNA and α7nAChR in synaptosomesprepared from frontal cortices of adult rats. Synaptosomes were exposedto 100 nM Aβ₄₂ for 30 minutes, and with 0.1 or 1 μM compounds addedeither simultaneously or 10 minutes earlier. Controls were a vehicle (noAβ₄₂) and an Aβ₄₂ alone condition.

FIG. 2 shows the Western blots from all nine compounds assayed, plus(+)naloxone (NLX), as well as the quantitation of the blots for the fourmost active compounds. All four of those compounds reduced theα7nAChR-FLNA association with 10-minute pre-incubation, and CompoundC0105 also markedly reduced this coupling with simultaneousadministration.

To assess Aβ₄₂ signaling via α7nAChR after compound administration,levels of phosphorylated ERK2 were measured in the same synaptosomepreparations treated with Aβ₄₂ and compounds. Phosphorylation of ERK2indicates its activation, which leads to tau phosphorylation. Comparedto the control condition, Aβ₄₂ strongly activates ERK2, and thisactivation is greatly suppressed by all four compounds at 0.1 and 1 nMwith 10 minute pretreatment, and also by Compound C0105 withsimultaneous treatment (FIG. 3).

Next assessed was whether the FLNA-binding compounds also decreasephosphorylation of tau, a downstream effect of Aβ₄₂ binding to α7nAChRand ERK2 activation. The three primary phosphorylation sites on tauprotein were examined for their phosphorylation levels compared to totaltau protein content. Tau, phosphorylated at these three sites, is aconstituent of NFTs. Consistent with effects on FLNA-α7nAChRassociation, and ERK2 activation, all of the FLNA-binding compoundsassayed, with 10 minute pre-incubation, decreased Aβ₄₂-inducedphosphorylation of tau at all three sites (FIG. 4).

Compounds C0105 and C0114 Decrease Binding of FITC-Aβ₄₂ to MembraneFractions Containing α7nAChR and FLNA

Using biotinylated α7nAChR-containing SK-N-MC cell fragments, compoundsand FITC-Aβ₄₂ were added simultaneously and incubated at 30° C. for 30minutes. Compound C0105 at 0.1, 1 and 10 nM concentration inhibitedFITC-Aβ₄₂ binding by 52.3±3.7%, 55.1±3.0%, and 56.5±4.2%, respectively.Compound C0114 was less effective, decreasing FITC-Aβ₄₂ binding by27.8±3.3%, 40.0±2.1%, and 53.4±3.6% at these three concentrations. Thesedata suggest that by binding to FLNA and likely changing itsconformation, Illustrative Compound C0105, and to a lesser extentCompound C0114, are able to affect the affinity of Aβ₄₂ binding toα7nAChR.

FLNA-α7nAChR/TLR4 and Aβ₄₂-α7nAChR Associations are Increased in ADLymphocytes

Because lymphocytes contain α7nAChR and TLR4, whether the associationsof these receptors with FLNA are increased in the lymphocytes of ADpatients and whether Compound C0105 treatment ex vivo could disruptthese associations were assessed. Also assayed was the effect ofCompound C0105 on Aβ₄₂-α7nAChR complexes in AD lymphocytes andAβ₄₂-treated lymphocytes from age-matched control subjects.

FLNA associations with α7nAChR and with TLR4 were dramatically increasedin AD and Aβ₄₂-treated control lymphocytes compared with vehicle-treatedcontrol lymphocytes. Additionally, the level of Aβ₄₂-α7nAChR complexeswas elevated in AD or Aβ₄₂-treated control lymphocytes. Incubation with1 nM Compound C0105 for 30 minutes significantly reduced the increasedassociations with FLNA and the level of Aβ₄₂-α7nAChR complexes.

Studies In Organotypic Frontocortical Brain Slice Cultures from AdultRats

Compounds C0105 and C0114 Reduce Aβ₄₂-Induced FLNA Association with Bothα7nAChR and TLR4

Organotypic frontocortical brain slices of adult rats were incubated for16 hours with 100 nM Aβ₄₂ and either illustrative Compound C0105 orC0114, added simultaneously. Tissue was harvested and solubilized, and aspecific antibody against FLNA was used to immunoprecipitate FLNA andassociated proteins. The FLNA immunoprecipitate was size-fractionatedusing SDS-PAGE, transferred and probed with specific antibodies directedagainst each of the following receptors: α7nAChR, TLR4, IR and MOR. Aβ₄₂(100 nM) caused an increase in FLNA association with both α7nAChR andTLR4, but not with IR or MOR (FIG. 5). Compound C0105 reduced theAβ₄₂-induced increase in FLNA-α7nAChR association at 0.1, 1 and 10 nM,and it reduced the increase in FLNA-TLR4 at 1 and 10 nM. Compound C0114,tested only at 1 and 10 nM, reduced the FLNA-α7AchR association at bothconcentrations and the FLNA-TLR4 association at 10 nM.

Compounds C0105 and C0114 Reduce Aβ₄₂-Induced Tau Phosphorylation

In organotypic frontocortical brain slice cultures treatedsimultaneously with Aβ₄₂ and either of Compounds C0105 or C0114, allthree concentrations of Compound C0105 and both concentrations ofCompound C0114 reduced tau phosphorylation at all three phosphorylationsites of tau found in neurofibrillary tangles (FIG. 6).

Compounds C0105 and C0114 Restore Impairment in α7nAChR Function Inducedby Aβ₄₂

Normal functioning of the α7nAChR is compromised by Aβ₄₂, as indicatedby reduced calcium influx after stimulating the receptor with a fullagonist (PNU282987;N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide). Both ofCompounds C0105 and C0114 restored functioning of this receptor, with 1and 10 nM of Compound C0105 fully restoring function (FIG. 7).

Compounds C0105 and C0114 Restore Impairment in NMDAR Function Inducedby Aβ₄₂

Similar to the assessment of α7nAChR function after Aβ₄₂ exposure, NMDARfunction was assessed by measuring calcium influx after stimulation withthe co-agonist, glycine and NMDA. The Aβ₄₂-induced impairment wascompletely prevented by 1 and 10 nM Compound C0105, with 0.1 nMcompounds C0105 and C0114 also significantly restoring NMDAR function(FIG. 8).

The NMDAR dysfunction was also assessed by measuring levels of sixdifferent signaling molecules (nNOS, PLCγ1, PKCγ, pY⁴⁰²PyK2, pY⁴¹⁶Src,and PSD-95) in association with the obligatory NMDAR subunit, NR1, afterstimulation with NMDA and glycine. NMDA with glycine intensified theassociation of NMDARs with PSD-95, increased the recruitment of nNOS,PLCγ1 and PKCγ to NMDAR complexes and elevated levels of active PyK2(pY⁴⁰²PyK2) and Src (pY⁴¹⁶Src) in the vehicle control condition; allthese elevations were severely compromised by Aβ₄₂ (FIG. 9). Both ofCompounds C0105 and C0114 fully restored function as evidenced by fullrestorations in the levels of these molecules with the receptor.

Compounds C0105 and C0114 Restore Impairment in IR Function Induced byAβ₄₂

A third receptor examined, the IR, also showed Aβ₄₂-induced dysfunction(FIG. 10). In the vehicle condition, after agonist (insulin) stimulationof the receptor, the association of the adaptor protein for downstreamsignaling molecule IRS-1 with IR (detected by immunoprecipitation withan antibody to IRβ) is increased, and the level of activated IR asindicated by the phosphorylated IRβ, pY^(1150/1151)IRβ. With Aβ₄₂exposure, levels of both the IRS-1 association and the pY^(1150/1151)IRβare dramatically reduced, indicating a desensitization of the receptoror a resistance to insulin. Despite observation that the IR-FLNAassociation is not affected by Aβ₄₂ nor by the compounds studied here,each of Compounds C0105 and C0114 normalized the signaling impairmentsinduced by Aβ₄₂ exposure.

Compounds C0105 and C0114 Prevent Aβ₄₂-Induced Cell Death as Indicatedby K⁺-Evoked Calcium Influx

One indication of non-functional or dying cells is their inability todepolarize and let in calcium. Hence, to measure cell death ornon-functionality in the slice cultures, calcium influx was measured inresponse to K⁺-evoked depolarization. Calcium influx was greatly reducedby Aβ₄₂, and this reduction was completely reversed by 1 and 10 nMcompound C0105 and by 10 nM compound C0114 (FIG. 11). The lowerconcentrations of compound C0105 (0.1 nM) and of Compound C0114 (1 nM)were also very effective in preventing cell death.

Compound C0105 Dramatically Reduces Immunostaining for NFT and Aβ₄₂Deposits

Immunohistochemistry using antibodies to pTau and Aβ₄₂, respectively,shows that administration of compound C0105 dramatically reduces bothNFT (FIG. 12) and Aβ₄₂ aggregates (FIG. 13) in organotypicfrontocortical brain slice cultures incubated with Aβ₄₂.

ICV Aβ₄₂ Infusion Mouse Model

In an intracerebroventricular (ICV) Aβ₄₂ infusion mouse model ofAlzheimer's disease, Aβ₄₂ dramatically increased FLNA association withboth α7nAChR and TLR4, caused tau phosphorylation at all threephosphorylation sites of tau found in neurofibrillary tangles, andimpaired signaling of α7nAChR, NMDAR and IR. Twice daily systemictreatment with 10 mg/kg of Compound C0105 markedly reduced these effectsof the Aβ₄₂ infusion. Compound C0105 reduced Aβ₄₂-induced increases inFLNA associations with both α7nAChR and TLR4 (FIG. 14), suggesting areduction in Aβ₄₂-mediated signaling of both these receptors.

Compound C0105 treatment suppressed tau phosphorylation at all threephosphorylation sites (FIG. 15), again indicating that Aβ₄₂ signalingvia α7nAChR is disrupted. The high-affinity binding of compound C0105 toFLNA appears to reduce Aβ₄₂ signaling via α7nAChR by reducing Aβ₄₂binding to α7nAChR: the level of these Aβ₄₂-α7nAChR complexes is reducedin C0105-treated animals (FIG. 16).

Aβ₄₂-induced dysfunction of α7nAChR was illustrated by calcium influxafter stimulation of α7nAChR with its full agonist PNU282987. TheAβ₄₂-induced reduction in this calcium influx was normalized in theCompound C0105-treated animals (FIG. 17). Likewise, downstream NMDARfunction was also impaired by ICV Aβ₄₂ infusion, illustrated by calciuminflux after co-stimulation with NMDA and glycine (FIG. 18). Again,Compound C0105 treatment restored the Aβ₄₂-induced NMDAR dysfunction.Calcium influx after depolarization by 1K⁺ was used to assess overallcellular dysfunction or dying cells. ICV Aβ₄₂ infusion greatly reducedthis K⁺-evoked calcium influx, and C0105 treatment restored it (FIG.19).

That Compound C0105 treatment can reverse NMDAR dysfunction is alsoevidenced by measuring NMDAR signaling. Aβ₄₂-infused mice showedreductions in NMDA/glycine-induced activation (phosphorylation) and inrecruitment to NMDAR of six signaling components. Compound C0105treatment of ICV Aβ₄₂-infused mice produced virtually identical results.

NMDAR signaling impairment was also assessed by levels of six signalingcomponents (PLCγ, nNOS pY⁴⁰²PyK2, PSD-95, PKCγ, pY⁴¹⁶Src, and NR1)co-immunoprecipitating with NR-1, the obligatory subunit of NMDAR, afterco-stimulating with NMDA and glycine. Aβ₄₂ reduced levels of all sixsignaling molecules, and these were normalized by C0105 treatment (FIG.20). Similarly, IR signaling was assessed by phosphorylation of IPβ andits association with the signaling molecule IRS-1 after stimulating withinsulin. Like NMDAR signaling, these measures were reduced by Aβ₄₂ andrestored by C0105 treatment (FIG. 21).

Importantly, because FLNA association with TLR4 is increased by Aβ₄₂ andnormalized by Compound C0105, whether the inflammatory cytokine releaseafter ICV 4342 infusion would be suppressed by Compound C0105 treatmentwas assessed. ICV Aβ₄₂ infusion did increase IL-6, TNF-α and IL-1βproduction. Compound C0105 treatment completely abolished theAβ₄₂-induced IL-6 production and suppressed TNF-α and IL-1β levels by 86and 80%, respectively (FIG. 22). Finally, immunohistochemistry (IHC) ofprefrontal cortex (FCX) and hippocampus (HP) shows that Compound C0105treatment prevents not only NFT formation (FIG. 23) but also amyloiddeposits (FIG. 24).

Postmortem Human Brain Tissue Study

In AD and Aβ₄₂-treated control tissue, Compound C0105 reducesAβ₄₂-induced FLNA association with α7nAChR and TLR4 (FIG. 25). Contactwith compound C0105 reduces Aβ₄₂-α7nAChR complexes (FIG. 26) by reducingthe affinity for this interaction about 1,000-10,000-fold (FIG. 27).Contact with Compound C0105 preserves functioning of α7nAChR (FIG. 28)and NMDAR (FIG. 29) as demonstrated by calcium influx after receptorstimulation. Normalization of receptor function was also demonstrated bylevels of signaling assemblies of NMDAR (FIG. 31) and IR (FIG. 32).Remarkably, one hour incubation of AD brain slices with 1 nM CompoundC0105 normalized all six measures of this NMDAR signaling impairmentwithout affecting the NMDAR subunit assemblies. Similar to findings inthe mouse model, Compound C0105 incubation also attenuated the deficitin depolarization-induced calcium influx in postmortem AD tissue thatindicates nonfunctioning or dying cells (FIG. 30).

In postmortem control frontocortical tissue, the FLNA pentapeptidebinding site of Compound C0105 (VAKGL of SEQ ID NO: 1) was used as adecoy to block the attenuation of FLNA association with α7nAChR and TLR4by Compound C0105 (FIG. 33). A similar blockade of the prevention of tauphosphorylation was demonstrated with this decoy pentapeptide (FIG. 34).Together these results confirm that Compound C0105 inhibits tauphosphorylation and preserves multiple receptor functions via itsbinding to FLNA.

Postmortem control frontocortical tissue was also used to demonstratethat Compound C0105 also reduces LPS-induced tau phosphorylation (FIG.47).

In postmortem control frontocortical tissue, Aβ₄₂-induced FLNArecruitment to α7nAChRs was mimicked by 10-fold higher concentrations ofAβ40 or of the α7nAChR agonists nicotine and PNU282987 withsignificantly lesser magnitudes. The α7nAChR antagonists α-bungarotoxinand methyllycaconitine and the cholinesterase inhibitor/positiveallosteric nicotinic receptor modulator galantamine showed no effect(Table, below). Interestingly, memantine, an antagonist of both NMDARand α7nAChR, elicited the recruitment of FLNA to α7nAChRs with amagnitude not far from that of Aβ40.

TABLE Effects of α7nAChR agents on FLNA recruitment to α7nAChRFLNA/α7nAChR ratio Statistics vs Aβ42 Statistics vs Agent (stimulationvs vehicle, %) vehicle, p effect, % Aβ42, p Vehicle 0.07 ± 0.01 — — —0.1 μM Aβ42 0.73 ± 0.05 (1012.3 ± 104.9) <0.0001 100 — 1 μM Aβ40 0. 28 ±0.03 (341.9 ± 65.1) 0.001 34.8 ± 6.6  <0.001 1 μM Nicotine 0.13 ± 0.02(107.7 ± 31.4) <0.001 11.0 ± 3.2  0.001 1 μM PNU282987 0.12 ± 0.02 (87.1± 37.9) 0.01 8.9 ± 3.9 0.001 1 μM α-Bungarotoxin 0.07 ± 0.01 (7.5 ± 6.6)0.65 0.8 ± 0.7 <0.001 1 μM Methyllycaconitine 0.08 ± 0.02 (19.8 ± 12.2)0.21 2.0 ± 1.2 <0.001 1 μM Galantamine 0.08 ± 0.02 (17.1 ± 14.6) 0.371.7 ± 1.5 <0.001 1 μM Memantine 0.21 ± 0.03 (225.9 ± 38.5) <0.00001 23.0± 3.9  0.001

Compounds

Compounds were synthesized and provided by Medicilon, Shanghai. Asidefrom the three syntheses described herein, more detailed syntheses areset out in one or more of US Patent Publications No. 2009/0191579 A1,No. 2010/0279996 A1, No. 2010/0279997 A1, No. 2010/0280061 A1, No.2011/0105481 A1, 2011/0105484 A1, No. 2011/0105487 A1, and No.2011/0105547 A1, whose disclosures are incorporated by reference.

A compound having an asymmetrical (chiral) carbon or a salt thereof canexist in the form of two enantiomers. The invention relates both to eachenantiomer and to their mixture; i.e., to both enantiomeric forms and totheir mixture. Additionally, where two or more chiral centers arepresent, diastereomers can form.

Where a contemplated compound or a pharmaceutically acceptable salt of acompound of Series A, B, C-1, C-2, D or E, or any of the other formulasherein is obtained in the form of a mixture of the stereoisomers,preferably in the form of the racemates or other mixtures of the variousenantiomers and/or diastereoisomers, they can be separated andoptionally isolated by conventional methods known to the person skilledin the art. Illustratively, for example, chromatographic separationprocesses are useful, particularly liquid chromatography processes understandard pressure or under elevated pressure, preferably MPLC and HPLCmethods, and also methods involving fractional crystallization. This canparticularly involve the separation of individual enantiomers, e.g.,diastereoisomeric salts separated by means of HPLC in the chiral phaseor by means of crystallization with chiral acids, for example(+)-tartaric acid, (−)-tartaric acid, or (+)-10-camphorsulfonic acid. Anenantiomer separated by chiral salt formation can readily be convertedinto an achiral or racemic pharmaceutically acceptable salt for use.

A compound of Series A, B, C-1, C-2, D or E or a pharmaceuticallyacceptable salt thereof is contemplated to be optionally used in aprocess of the invention in enantiomerically pure form; i.e., in (S) or(R) configuration or d and 1 forms, or in the form of a racemic mixtureshowing an (S,R) or (d,l) configuration, or as one or morediastereomers, and mixtures thereof.

Thus, a contemplated compound or its pharmaceutically acceptable saltcan optionally be present in one or more forms. Illustratively, thecompound or its salt can be in the form of an individual enantiomer ordiastereoisomer. A contemplated compound or its salt can also be presentin the form of a mixture of stereoisomers. A contemplated compound orsalt can also be present in the form of a racemic mixture.

A compound useful as an active ingredient in a contemplated method canbe readily synthesized. An illustrative synthetic scheme (Scheme 1) isshown below for the compounds of Series A. Similar schemes are set outthereafter for the preferred compound types.

Similar syntheses can be carried out for phenolic compounds, startingwith phenol or a substituted phenol in place of D-menthol that is shownin Scheme 1. Another cyclohexanol or cyclohexenol can also be used inplace of D-menthol. The alcohol formed by reaction of Compound 1 with anamine can be readily oxidized by known methods.

Table of Series-A Compounds

A3333

A0001

A0002

A0003

AOOO4

A0005

A0006

A0007

A0008

AOOO9

A0010

A0011

A0012

A0013

A0014

A0015

A0017

A0020

A0021

A0022

A0025

A0026

A0028

A0029

A0030

A0031

A0032-1

A0032

A0033

A0035

A0036

A0037

A0038

A0039

A0040

A0041

A0042

A0043

A0044

A0045

A0046

A0047

A0048

A0049

A0050

A0051

A0053

A0054

A0055

A0056

A0057

A0058

A0059

A0060

A0061

A0068

A0075

A0076

A0077

A0078

A compound of Series B can be prepared by following the synthetic routeillustrated in Scheme 2, below. An illustrative synthetic scheme isshown below for the preparation of a first portion of a contemplatedcompound, with the second portion being added by a reaction with anappropriately substituted methylketone compound in the presence of astrong base such as sodium ethoxide. The resulting ketone can beconverted into the corresponding alcohol by mild reduction as withsodium borohydride. A ketone or alcohol can be converted to thequaternary nitrogen atom-containing compound using an alkylating agentsuch as methyl iodide.

Table of Series-B Compounds

B0001

B0002

B0004

B0005

B0006

B0007

B0008

B0011

B0012

B0015

B0016

B0017

B0018

B0019

B0020

B0021

B0023

B0024

B0025

B0026

B0027

B0028

B0029

B0030

B0031

B0032

B0033

B0034

B0035

B0036

B0037

B0038

BOO39

B0040

B0041

BOO42

B0043

B0044

B0045

B0047

B0048

B0049

B0050

B0051

B0052

B0053

B0055

B0056

B0057

B0058

B 0059

B0060

B0061

B0062

B0063

B0064

B0065

B0067

B0068

5009

6810

An illustrative synthetic scheme is shown below for preparation ofSeries-C (both C-1 and C-2) compounds that contain various substituentsand ring atoms. That scheme can be readily adapted for the preparationof compounds containing two carbonyl linkages, and also one carbonyl andone sulfonyl linkage in the opposite configurations from those shown.Ethanolamine or thioethanolamine can be replaced by ethylenediamine orN-methylethylene-diamine to prepare the corresponding dinitrogencompounds. Similar replacement with 2-aminoacetamide or an N-substitutedacetamide or propionamide provides the correspondingaminoamido-containing ring system.

Table of Series-C-1 Compounds

7866

C0001

C0002

C0003

C0004

C0005

C0006

C0007

C0008

C0009

C0010

C0011

C0012

C0013

C0014

C0015

C0016

C0017

C0018

C0019

C0021

C0022

C0023

C0024

C0025

C0026

C0027-1

C0028

C0029

C0030

C0031

C0032

C0033

C0034

C0034-3

C0037-2

C0038

C0040

C0041

C0042

C0044

C0045

C0047

C0048

C0049

C0049-2

C0050

C0051

C0052

C0053

C0054

C0055-4

C0055

C0056

C0057

C0058

C0059

C0060

C0061

C0062

C0064

C0065

C0066

C0067

C0068

C0068-2

C0069

C0070

C0071

C0071-2

C0072

C0073

C0077

C0078

C0078-2

C0080

C0082M

C0083M

C0084M

C0085M

C0087M

C0136M(P5)

C0138M

C0139M

C0140M

C0141M

C0141M-2

C0142M

C0143M-2

C0143M

C0143M-2

C0144M

C0144M-2

C0145M

C0146M

C0147M A2

C0148M

C0149M-2

C0149M

C0150M

C0151M

C0151M-2

C0152M-4

Table of Series C-2 Compounds

S-C0027

C0027

C0043

C0046

C0053-3

C0079M-7

C0080M-6

C0081M-7

C0086M

C0088M

C0089M

C0090M

C0091M

C0092M

C0093M

C0094M

C0095M

C0096M

C0097M

C0099M

C0100M

C0101M

C0102M

C0104M

C0105 M

C0106 M

C0108M

C0109M

C0111 M

C0114M

C0115M

C0116M

C0118M

C0119M

C0123 M

C0124M

C0125M

C0126 M

C0128M

C0129M

C0133M

C0134M

F-C0134

C0135M

C0137M P7

C0145M-3

C0153M-3

Compound 4

Compound 9

Compound 10

Table of Series D Compounds

Compound A

Compound B

Compound C

Preparation of Series C-2 Compounds 4, 9 and 10

These three compounds were prepared via a common intermediate designated9-2 herein that was prepared during the synthesis of Compound C0116M inapplication Ser. No. 12/5610,091 (US Publication No. 20110105487 A1dated May 5, 2011; WO 2010/051497), and referred to therein as CompoundC0116M-1.

After preparation of Compound 9-2, the syntheses of Compounds 9 and 10proceeded routinely by first adding the tosyl group in pyridine to thenitrogen of the five-membered ring, followed by removal of the t-BOCgroup with trifluoroacetic acid (TFA) in dichloromethane to formCompound 9-4 as shown below. Specifics of the syntheses are providedhereinafter.

Compound 9-2 also served as the basis for preparation of Compound 4.Here, as shown below, 4-trifluoromethoxyphenyl sulfonylchloride wasreacted in pyridine with the amine of the five-membered ring, and thet-BOC group removed in TFA/DCM as above to form Compound 4-1. The aminenitrogen of the six-membered ring of Compound 4-1 was then reacted with(bromomethyl)cyclopropane to form the N-alkylated product that isCompound 4.

Preparation of Compound 9-2

To a solution of N-Boc-piperidin-4-one (50 g, 251 mmol) in ethanol (500mL) was added 2-aminoethanol (46 g). The mixture was stirred at roomtemperature overnight (about 18 hours). Then the solvent was removedunder reduced pressure. The residue was diluted with CH₂Cl₂ (DCM) andwashed with saturated aqueous Na₂CO₃ (100 mL×6). The organic phase wasdried over anhydrous Na₂SO₄, and concentrated to provide the product asa yellow oil (61 g, yield: 100%, confirmed by TLC).

Preparation of Compound 9-3

Toluenesulfonyl chloride (TsCl; 24.7 g, 130 mmol) was added to asolution of Compound 9-2 (31.2 g, 130 mmol) in pyridine (320 mL). Themixture was stirred overnight (about 18 hours) at room temperature. Thereaction mixture was concentrated in vacuo to remove the pyridine andthe residue was dissolved with DCM and washed with saturated NaHCO₃. Theorganic layer was dried over Na₂SO₄, concentrated and purified by columnchromatography to provide the product as white solid (40 g, yield: 78%,confirmed by ¹H NMR).

¹H NMR (400 MHz, CDCl₃): 7.74 (d, J=8.4 Hz, 2H); 7.31 (d, J=8.0 Hz, 2H);4.13-4.03 (m, 4H); 3.56˜3.50 (m, 2H); 2.89 (brs, 2H); 2.46 (s, 3H);2.43˜2.36 (m, 2H); 1.63 (brs, 2H); 1.47 (s, 9H).

Preparation of Compound 9-4

Trifluoroacetic acid (CF₃COOH; 60 mL) was added to a solution ofCompound 9-3 (35.2 g, 88.7 mmol) in DCM (350 mL). The mixture wasstirred at ice/water for 50 minutes. To the reaction mixture was added200 mL of DCM, and the resulting composition washed with saturatedNa₂CO₃. The organic layer was dried over Na₂SO₄, concentrated in vacuoto provide the crude product. The crude product was purified by columnchromatography to provide the desired product as pale yellow oil (11.2g, yield: 42%, confirmed by ¹H NMR).

¹H NMR (400 MHz, CDCl₃): 7.75 (d, J=8.4 Hz, 2H); 7.29 (d, J=8.0 Hz, 2H);3.95 (t, J=6.4 Hz, 2H); 3.75 (brs, 1H); 3.51˜3.48 (t, J=5.6 Hz, 2H);3.16˜3.12 (dd, J=12.4, 4.0 Hz, 2H); 2.92˜2.86 (td, J=12.8, 2.0 Hz, 2H);2.48˜2.44 (m, 2H); 2.41 (s, 3H); 1.65 (d, J=12.8 Hz, 2H).

Preparation of Compound 9

(Bromomethyl)cyclobutane (1.86 g, 12.5 mmol) was added to a mixture ofCompound 9-4 (1.85 g, 6.25 mmol) and K₂CO₃ (3.39 g, 12.5 mmol) inacetone (40 mL), and the reaction mixture was stirred at refluxovernight (about 18 hours). After cooling, the mixture was filtered andconcentrated, purified by chromatography with ethyl acetate (EA) toobtain crude product as pale yellow solid (1.6 g, yield: 70%, confirmedby LCMS, ¹H NMR showed it was impure). The crude product was purified byfurther chromatography with EA to provide the desired product as whitesolid (1.15 g, yield: 50%, confirmed by LCMS and ¹H NMR, HPLC: 99.3% @254 nm, 99.5 @ 214 nm).

¹H NMR (400 MHz, CDCl₃): 7.77 (d, J=8.0 Hz, 2H); 7.31 (d, J=8.0 Hz, 2H);3.95 (t, J=6.0 Hz, 2H); 3.52 (t, J=6.4 Hz, 2H); 2.76˜2.73 (d, J=10.0 Hz,2H); 2.54˜2.39 (m, 8H); 2.21˜2.15 (t, J=11.6 Hz, 2H); 2.07˜2.05 (m, 2H);1.93˜1.88 (m, 2H); 1.70˜1.65 (m, 2H); 1.56˜1.53 (d, J=12.4 Hz, 2H). MS(ESI) calcd for C₁₉H₂₈N₂O₃S (m/z): 364.18. found: 365.1 [M+1]⁺.

Preparation of Compound 10

To a mixture of Compound 9-4 (1.72 g, 5.8 mmol) and K₂CO₃ (1.6 g, 11.6mmol) in acetone (30 mL) was added 3-bromoprop-1-ene (0.7 g, 5.8 mmol),and the reaction mixture was stirred at 40° C. for 2 hours. Aftercooling, the mixture was filtered and concentrated, purified bychromatography with EA to obtasin the desired product as white solid(1.1 g, 56% yield, confirmed by LCMS and ¹H NMR, HPLC: 98.8% @ 254 nm,98.9 @ 214 nm).

¹H NMR (400 MHz, CDCl₃): 7.76˜7.74 (d, J=8.0 Hz, 2H); 7.29˜7.27 (d,J=8.0 Hz, 2H); 5.90˜5.82 (m, 1H); 5.18˜5.11 (m, 2H); 3.95˜3.91 (t, J=6.0Hz, 2H); 3.52˜3.49 (t, J=6.0 Hz, 2H); 3.0˜2.98 (d, J=6.4 Hz, 2H);2.83˜2.80 (dd, J=8.8, 2.4 Hz, 2H); 2.55˜2.48 (td, J=13.2, 4.4 Hz, 2H);2.41 (s, 3H); 2.19˜2.14 (t, J=11.2 Hz, 2H); 1.58˜1.55 (d, J=12.0 Hz,2H). MS (ESI) calcd for C₁₇H₂₄N₂O₃S (m/z): 336.45. found: 337.1 [M+1]

Preparation of Compound 11

To a solution of Compound 9-2 (14.6 g, 60 mmol) in pyridine (150 mL) wasadded 4-(trifluoro-methoxy)benzene-1-sulfonyl chloride (15.7 g, 60mmol). The mixture was stirred overnight (about 18 hours) at roomtemperature. The reaction mixture was concentrated in vacuo to removethe pyridine and the residue was dissolved with DCM, washed withsaturated NaHCO₃. The organic layer was dried over Na₂SO₄, concentratedand purified by column chromatography to give product as white solid (20g, yield: 71%, confirmed by ¹H NMR and LCMS).

¹H NMR (400 MHz, CDCl₃): 7.92˜7.90 (d, J=8.4 Hz, 2H); 7.35˜7.32 (d,J=8.4 Hz, 2H); 4.13˜3.97 (m, 4H); 3.51 (brs, 2H); 2.90 (brs, 2H);2.45˜2.35 (m, 2H); 1.58 (brs, 2H); 1.47 (s, 9H). MS (ESI) calcd forC₁₉H₂₅F₃N₂O₆S (m/z): 466.14. found: 367.0 [M+1]⁺.

Preparation of Compound 4-1

Trifluoroacetic acid (CF₃COOH; 20 mL) was added to a solution ofCompound 11 (15 g, 32 mmol) in DCM (150 mL). The mixture was stirred atice/water for 50 minutes. The reaction mixture was added to 200 mL ofDCM, washed with saturated Na₂CO₃. The organic layer was dried overNa₂SO₄, concentrated in vacuo to provide the crude product. The crudeproduct was purified by column chromatography to provide the desiredproduct as pale yellow solid (5.9 g, yield: 50%, confirmed by ¹H NMR).

¹H NMR (400 MHz, CDCl₃): 7.93˜7.91 (d, J=8.4 Hz, 2H); 7.34˜7.32 (d,J=8.0 Hz, 2H); 4.0˜3.97 (t, J=5.6 Hz, 2H); 3.51˜3.48 (t, J=6.0 Hz, 2H);3.06˜3.02 (dd, J=12.4, 4.0 Hz, 2H); 2.86˜2.80 (t, J=13.2 Hz, 2H);2.39˜2.31 (td, J=12.8, 4.8 Hz, 2H); 2.48˜2.44 (m, 2H); 1.64˜1.62 (d,J=12.8 Hz, 2H).

Preparation of Compound 4

(Bromomethyl)cyclobutane (1.5 g, 4.1 mmol) was added to a mixture ofCompound 4-1 (1.5 g, 4.1 mmol) and K₂CO₃ (1.13 g, 8.2 mmol) in acetone(15 mL), and the reaction mixture was stirred at reflux for 4 hours.After cooling, the mixture was filtered and concentrated, purified bychromatography with EA to provide the desired product as an off-whitesolid (1.05 g, yield: 61%, confirmed by LCMS and ¹H NMR, HPLC: 96.9% @254 nm, 98.4 @ 214 nm).

¹H NMR (400 MHz, CDCl₃): 7.91˜7.94 (d, J=8.8 Hz, 2H); 7.34˜7.32 (d,J=8.0 Hz, 2H); 3.98˜3.95 (t, J=6.0 Hz, 2H); 3.54˜3.51 (t, J=6.4 Hz, 2H);2.99˜2.96 (dd, J=8.8, 2.0 Hz, 2H); 2.56˜2.49 (td, J=12.8, 4.4 Hz, 2H);2.26˜2.17 (m, 4H); 1.60˜1.57 (d, J=12.4 Hz, 2H); 0.87˜0.84 (m, 1H);0.52˜0.48 (m, 2H); 0.10˜0.07 (m, 2H). MS (ESI) calcd for C₁₈H₂₃F₃N₂O₄S(m/z): 420.13. found: 421.1 [M+1]⁺.

Preparation of Series D Compounds A, B and C

Compound 4

To a solution of Compound 1 (10 g, 57 mmol) in THF (100 mL) was added1,1′-carbonyldiimidazole (CDI) (11.1 g, 68.5 mmol) at room temperature,and the mixture was stirred for 30 minutes. Compound 2 (7.34 g, 68.5mmol) was then added and stirred overnight (about 18 hours). The solventwas evaporated and the residue was dissolved in ethyl acetate (EA; 400mL) to which was added 4M HCl/MeOH (50 mL), and the resulting admixturewas stirred overnight (about 18 hours). The resulting white solid wasfiltered and suspended in EA, washed with aq.NaHCO₃ and concentrated toafford product as white solid (3.2 g, 34% yield, as confirmed by NMR).

1H-NMR (400 MHz, CDCl₃): 3.41 (s, 3H); 4.48 (d, J=6.0 Hz, 2H); 7.26˜7.36(m, 5H); 7.57 (br, s, 1H).

Compound A-3

A mixture of Compound A-1 (3.75 g, 24 mmol), A-2 (1.5 g, 11 mmol) andtriethylamine (TEA) (4.5 g, 44.38 mmol) in dichloromethane (DCM) (50 mL)was stirred at room temperature overnight (about 18 hours). The reactionmixture was washed with water, dried over Na₂SO₄ and concentrated toafford product as white solid (2.55 g, 98% yield, confirmed by NMR).

¹H-NMR (400 MHz, CDCl₃): 2.53 (t, J=6.4 Hz, 4H); 4.01 (t, J=6.4 Hz, 4H);7.10-7.30 (m, 5H).

Compound A

A mixture of Compound A-3 (400 mg, 1.7 mmol) and Compound 4 (280 mg, 1.7mmol) in methanol (60 mL) was heated to reflux overnight (about 18hours) under argon. The mixture was concentrated and purified by pre-TLCto get product as pale white solid (84 mg, 13% yield, NMR and MSconfirmed, 98% by HPLC).

¹H-NMR (400 MHz, CDCl₃): 1.42 (d, J=12.4 Hz, 2H); 1.92 (dt, J=4.4, 13.2Hz, 2H); 3.32 (dt, J=2.0, 12.8 Hz, 2H); 3.52 (s, 2H); 4.42 (s, 2H); 4.47(s, 2H); 7.06 (t, J=7.6 Hz, 2H); 7.14 (t, J=7.6 Hz, 1H); 7.24-7.33 (m,9H). MS (ESI) calcd for C₂₁H₂₄N₄OS (m/z): 380.17. found: 381.2 [M+1]⁺.

Compound B-4

To a solution of pyridin-4-amine (400 mg, 4.25 mmol) in THF (35 mL) wasadded 60% NaH (340 mg, 8.5 mmol) in an ice bath, and the mixture wasstirred for 1 hour. Compound B-2 (0.99 g, 4.25 mmol) was added and themixture was permitted to gradually to reach room temperature and stirredfor 3 hours. Compound A-1 (0.78 g, 5.1 mmol) andN,N-diisopropyl-ethylamine (DIEA; 1 mL) was added and the mixture wasstirred at room temperature overnight (about 18 hours). Water was addedand the resulting composition was extracted with EA, washed with brine,dried over Na₂SO₄, concentrated and purified by column chromatography toafford oil (0.23 g, 23% yield, NMR was not pure but the major componentwas title compound).

¹H-NMR (400 MHz, CDCl₃): 2.66 (t, J=6.4 Hz, 4H); 4.12 (t, J=6.4 Hz, 4H);7.11-7.12 (d, J=4.8 Hz, 2H); 8.50˜8.52 (d, J=5.6 Hz, 2H).

Compound B

A solution of Compound B-4 (230 mg, 1.7 mmol) and Compound 4 (225 mg,1.37 mmol) in methanol (25 mL) was heated to reflux overnight (about 18hours) under argon. The mixture was concentrated and purified by pre-TLCto get product as yellow solid (45 mg, 12% yield, NMR and MS confirmed,96% by HPLC).

¹H-NMR (400 MHz, CDCl₃): 1.47 (d, J=13.2 Hz, 2H); 1.92˜1.98 (m, 2H);3.41 (t, J=13.2 Hz, 2H); 3.54 (s, 2H); 4.44 (s, 4H); 6.95 (d, J=4.4 Hz,2H); 7.26˜7.31 (m, 5H); 8.45 (d, J=4.0 Hz, 2H); MS (ESI) calcd forC₂₀H₂₃N₅OS (m/z): 381.16. found: 382.4 [M+1]⁺.

Compound C-2

A mixture of Compound C-1 (1.0 g, 8.39 mmol), Compound A-1 (2.83 g,18.45 mmol) and potassium carbonate (4.64 g, 33.6 mmol) in DCM (50 mL)was stirred at ambient temperature for 18 hours. The mixture was washedwith water, 1N HCl (aqueous), dried over Na₂SO₄ and concentrated. Theresidue was purified by column chromatography (PE/EA=3:1) to affordproduct as white solid (1.0 g, 55% yield, NMR confirmed the titlecompound).

¹H-NMR (400 MHz, CDCl₃): 2.56 (t, J=6.4 Hz, 4H); 3.81 (t, J=6.4 Hz, 4H);6.50 (brs, 1H); 7.07 (t, J=7.2 Hz, 4H); 7.28-7.37 (m, 4H).

Compound C

A mixture of Compound C-2 (400 mg, 1.84 mmol) and Compound 4 (400 mg,2.44 mmol) in methanol (40 mL) was heated to reflux overnight (about 18hours) under argon. The mixture was concentrated and purified by pre-TLCto provide the product as white solid (96 mg, 14% yield, NMR and MSconfirmed, 98% by HPLC).

¹H-NMR (400 MHz, CDCl₃): 1.45 (d, J=12.0 Hz, 2H); 1.85 (dt, J=4.4, 13.2Hz, 2H); 3.19 (dt, J=2.0, 13.2 Hz, 2H); 3.55 (s, 2H); 3.96 (dt, J 13.6,2.0 Hz, 2H); 4.44 (s, 2H); 6.29 (s, 1H); 7.02˜7.06 (m, 1H); 7.22˜7.33(m, 10H); MS (ESI) calcd for C₂₁H₂₄N₄O₂ (m/z): 364.19. found: 365.2[M+1]⁺.

Example 1 FITC-NLX-based FLNA Screening Assay

A. Streptavidin-Coated 96-Well Plates

Streptavidin-coated 96-well plates (Reacti-Bind™ NeutrAvidin™ Highbinding capacity coated 96-well plate, Pierce-ENDOGEN) are washed threetimes with 200 μl of 50 mM Tris HCl, pH 7.4 according to themanufacturer's recommendation.

B. N-Biotinylated VAKGL Pentapeptide (Bn-VAKGL) (SEQ ID NO: 1)

Bn-VAKGL peptide (0.5 mg/plate) is dissolved in 50 μl DMSO and thenadded to 4450 μl of 50 mM Tris HCl, pH 7.4, containing 100 mM NaCl andprotease inhibitors (binding medium) as well as 500 μl superblock in PBS(Pierce-ENDOGEN) [final concentration for DMSO: 1%].

C. Coupling of Bn-VAKGL Peptides to Streptavidin-Coated Plate

The washed streptavidin-coated plates are contacted with 5 μg/well ofBn-VAKGL (100 μl) for 1 hour (incubated) with constant shaking at 25° C.[50 μl of Bn-VAKGL peptide solution from B+50 μl binding medium, finalconcentration for DMSO: 0.5%]. At the end of the incubation, the plateis washed three times with 200 μl of ice-cold 50 mM Tris HCl, pH 7.4.

D. Binding of FITC-Tagged Naloxone

[FITC-NLX] to VAKGL

Bn-VAKGL coated streptavidin plates are incubated with 10 nM fluoresceinisothiocyanate-labeled naloxone (FITC-NLX; Invitrogen) in binding medium(50 mM Tris HCl, pH 7.4 containing 100 mM NaCl and protease inhibitors)for 30 minutes at 30° C. with constant shaking. The final assay volumeis 100 μl. At the end of incubation, the plate is washed twice with 100μl of ice-cold 50 mM Tris, pH 7.4. The signal, bound-FITC-NLX isdetected using a DTX-880 multi-mode plate reader (Beckman).

E. Screening of Medicinal Chemistry Analogs

The compounds are first individually dissolved in 25% DMSO containing 50mM Tris HCl, pH 7.4, to a final concentration of 1 mM (assisted bysonication when necessary) and then plated into 96-well compound plates.To screen the medicinal chemistry analogs (new compounds), each compoundsolution (1 μl) is added to the Bn-VAKGL coated streptavidin plate with50 μl/well of binding medium followed immediately with addition of 50 μlof FITC-NLX (total assay volume/well is 100 μl). The final screeningconcentration for each new compound is initially 10 M.

Each screening plate includes vehicle control (total binding) as well asnaloxone (NLX) and/or naltrexone (NTX) as positive controls. Compoundsare tested in triplicate or quadruplicate. Percent inhibition ofFITC-NLX binding for each compound is calculated [(Total FITC-NLX boundin vehicle−FITC-NLX bound with compound)/Total FITC-NLX bound invehicle]×100%]. To assess the efficacies and potencies of the selectedcompounds, compounds that achieve approximately 60-70% inhibition at 10μM are screened further at 1 and 0.1 μM concentrations.

The results of this screening assay are shown in the tables below.

FLNA Peptide Binding Assays A-Series Compounds

The R¹, R², R⁷ and R⁸, A, B and X groups are defined elsewhere herein.

Concentration of FLNA-binding Compound FLNA-binding 0.01 μM 0.1 μM 1 μMCompound Percent Binding Inhibition Naloxone 39.87% 46.29% 50.91%Control Average 3333 40.4% 48.5% 54.2% A0001 39.7% 45.6% 52.4% A000238.7% 43.7% 49.9% A0003 21.3% 31.6% 37.4% A0004 40.0% 43.7% 47.6% A000534.2% 38.2% 43.8% A0006 37.9% 43.5% 47.5% A0007 39.2% 46.2% 52.9% A000834.5% 33.5% 39.8% A0009 26.4% 37.8% 38.9% A0010 36.0% 36.5% 39.0% A001145.7% 51.1% 52.8% A0012 39.7% 49.6% 54.4% A0013 30.2% 40.2% 47.7% A001433.8% 39.7% 44.7% A0015 36.3% 46.8% 55.0% A0017 29.8% 38.6% 44.0% A002037.8% 38.8% 45.8% A0021 36.8% 43.4% 49.5% A0022 41.9% 49.7% 56.8% A002539.0% 49.8% 53.2% A0026 36.4% 42.4% 49.2% A0028 39.5% 43.8% 50.5% A002944.4% 44.4% 50.8% A0030 35.6% 44.4% 48.9% A0031 40.8% 47.6% 52.9%A0032-1 35.6% 43.9% 50.0% A0032 43.0% 50.3% 54.5% A0033 46.4% 51.8%56.5% A0035 40.3% 45.5% 54.9% A0036 45.6% 50.1% 54.4% A0037 49.3% 51.3%56.8% A0038 46.4% 52.3% 56.6% A0039 49.0% 53.5% 60.3% A0040 45.0% 50.4%56.3% A0041 45.8% 51.7% 56.9% A0042 47.2% 48.3% 55.8% AOO43 46.4% 48.9%51.8% A0044 32.4% 36.9% 39.6% A0045 28.1% 35.0% 37.8% A0046 34.3% 38.4%40.9% A0047 40.9% 42.9% 44.5% A0048 38.5% 44.0% 46.9% A0049 46.2% 49.4%49.3% A0050 42.9% 49.8% 52.1% A0051 45.9% 45.4% 52.1% A0053 34.8% 40.0%46.9% A0054 28.7% 35.8% 41.4% A0055 28.1% 32.4% 41.8% A0056 34.4% 40.9%41.3% A0057 29.1% 37.0% 43.4% A0058 28.9% 36.6% 42.1% A0059 27.4% 36.6%38.7% A0060 32.4% 39.0% 42.0% A0061 27.5% 38.9% 42.8% A0062 — — — A006321.2% 31.0% 38.8% A0064 41.8% 46.2% 53.6% A0065 38.7% 50.0% 50.8% A006636.7% 45.4% 53.7% A0067 32.7% 39.1% 44.3% A0068 51.9% 54.2% 58.3% A006932.0% 40.4% 46.1% A0070 32.9% 39.1% 41.7% A0071 44.7% 46.8% 53.9% A007245.5% 52.2% 59.4% A0073 47.3% 54.8% 59.7% A0074 — — — A0075 — — — A007636.1% 40.0% 44.9% A0077 41.1% 48.7% 49.4% A0078 50.1% 55.8% 57.6%Naloxone 39.87% 46.29% 50.91% Control Average

B-Series Compounds

The R¹, R², R³ and R⁴, W, X⁻ and Z groups, the dashed line, n and m aredefined elsewhere herein.

Concentration of FLNA-binding Compound FLNA-binding 0.01 μM 0.1 μM 1 μMCompound Percent Binding Inhibition Naloxone 39.87% 46.29% 50.91%Control Average 5009 42.5% 47.3% 54.3% B0001 37.1% 48.8% 54.3% B000240.2% 46.4% 55.0% B0003 45.4% 52.9% 63.5% B0004 38.9% 50.0% 54.8% B000531.8% 34.8% 41.7% B0006 45.1% 53.5% 61.3% B0007 43.6% 53.1% 57.3% B000835.5% 40.3% 52.8% B0009 39.6% 47.6% 53.6% B0010 39.4% 43.4% 50.3% B001140.9% 50.3% 55.8% B0012 39.4% 46.9% 51.7% B0013 25.2% 35.1% 43.4% B001425.7% 30.9% 37.8% B0015 30.4% 35.3% 42.3% B0016 27.1% 33.7% 41.9% B001728.3% 36.6% 44.6% B0018 37.2% 43.7% 47.6% B0019 34.3% 41.0% 49.0% B002038.1% 45.5% 50.6% B0021 32.5% 43.1% 47.6% B0022 34.3% 40.4% 45.6% B002328.5% 37.8% 46.4% B0024 34.8% 43.4% 47.7% B0025 41.7% 49.4% 56.6% B002641.1% 43.3% 48.2% B0027 40.2% 46.7% 49.8% B0028 38.2% 42.8% 49.1% B002933.4% 42.9% 50.2% B0030 47.0% 50.5% 57.6% B0031 36.2% 44.2% 50.5% B003245.1% 51.3% 48.9% B0033 42.1% 46.8% 49.4% B0034 49.1% 54.2% 59.1% B003545.4% 44.7% 51.0% B0036 46.6% 52.8% 62.1% B0037 47.4% 53.0% 52.4% B003841.2% 50.1% 57.0% B0039 43.3% 45.7% 50.9% B0040 40.0% 53.1% 57.1% B004144.0% 46.8% 52.8% B0042 40.8% 46.4% 51.6% B0043 30.8% 39.2% 46.8% B004435.2% 39.5% 44.4% B0045 63.2% 68.2% 73.9% B0046 42.2% 50.2% 55.4% B004730.7% 37.6% 47.1% B0048 34.7% 41.9% 43.9% B0049 32.2% 40.1% 47.1% B005029.2% 34.5% 39.8% B0051 29.9% 35.7% 43.7% B0052 30.2% 39.1% 44.3% B005333.1% 37.3% 47.6% B0054 25.6% 32.6% 43.3% B0055 63.2% 68.2% 73.9%Naloxone 39.87% 46.29% 50.91% Control Average

C-Series-1 Compounds

Each designation in the above formula is defined elsewhere herein.

Concentration of FLNA-binding Compound FLNA-binding 0.01 μM 0.1 μM 1 μMCompound Percent Binding Inhibition Naloxone 39.87% 46.29% 50.91%Control Average 7866 38.5% 47.9% 53.4% C0001 34.8% 42.9% 51.3% C000238.4% 45.6% 42.8% C0003 38.3% 45.3% 48.8% C0004 37.6% 42.3% 44.7% C000535.2% 44.5% 51.5% C0006 41.6% 46.8% 51.8% C0007 40.5% 46.3% 48.9% C000842.2% 52.3% 54.4% C0009 41.7% 49.0% 53.9% C0010 39.8% 42.7% 47.1% C001137.6% 41.4% 46.0% C0012 26.3% 39.5% 46.4% C0013 39.6% 42.4% 49.1% C001429.5% 38.8% 40.0% C0015 31.2% 40.6% 45.5% C0016 38.3% 43.8% 49.1% C001728.9% 35.4% 40.7% C0018 42.3% 45.9% 53.4% C0019 30.1% 38.2% 43.6% C002134.0% 38.4% 40.6% C0022 34.5% 37.6% 43.9% C0023 35.9% 41.7% 47.2% C002437.9% 46.4% 50.4% C0025 37.2% 41.4% 45.1% C0028 32.2% 36.6% 43.3% C002938.6% 43.2% 50.5% C0030 37.4% 45.4% 56.0% C0032 41.5% 50.5% 55.3% C003343.9% 48.4% 51.3% C0034 29.6% 38.3% 44.8% C0038 31.7% 36.0% 43.5% C004138.3% 47.0% 51.2% C0042 42.4% 49.7% 56.1% C0047 30.8% 35.2% 41.4% C004828.5% 38.9% 45.9% C0049 25.3% 27.9% 30.3% C0051 27.0% 30.4% 36.4% C005228.0% 35.6% 40.8% C0053 28.9% 33.8% 39.3% C0054 32.9% 39.4% 43.3% C0057 ND* ND ND C0060 60.3% 64.0% 68.0% C0061 ND ND ND C0062 39.5% 49.5%48.0% C0064 37.3% 44.4% 49.2% C0065 37.1% 44.0% 47.0% C0067 31.3% 39.7%45.0% C0068 53.7% 58.6% 62.2% C0069 ND ND ND C0070 42.6% 50.6% 53.6%C0071 39.1% 49.6% 55.2% C0072 28.4% 37.4% 44.0% C0073 ND ND ND C007745.7% 47.7% 51.0% C0078 46.6% 48.0% 50.5% C0080M 46.8% 53.3% 54.6%C0084M 47.2% 53.7% 55.9% C0085M 45.7% 53.7% 60.7% C0138M 53.0% 52.0%59.5% C0139M 48.9% 53.1% 61.6% C0140M 42.3% 49.2% 54.4% C0141M 33.1%39.0% 46.9% C0143M 45.3% 48.4% 57.8% C0144M 46.4% 50.7% 55.7% C0145M45.1% 53.7% 58.3% C0148M 46.2% 52.0% 57.0% C0149M 48.5% 52.3% 62.0%C0150M 47.3% 51.8% 61.4% C0151M 48.3% 51.7% 58.7% C0152M ND ND ND C0154MND ND ND Naloxone 39.87% 46.29% 50.91% Control Average *ND = Not Done.

C-Series-2 Compounds

Each designation in the above formula is defined elsewhere herein.

Concentration of FLNA-binding Compound FLNA-binding 0.01 μM 0.1 μM 1 μMCompound Percent Binding Inhibition Naloxone 39.87 46.29% 50.91 ControlAverage C0011 37.6% 41.4% 46.0% C0026 42.3% 44.8% 49.0% C0027 50.8%61.2% 63.8% S-C0027 39.1% 46.5% 53.6% C0034-3 29.6% 38.3% 44.8% C0037-2 ND* ND ND C0040 38.4% 46.3% 55.9% C0043 43.9% 51.3% 58.0% C0044 37.3%43.9% 50.6% C0045 39.1% 48.9% 53.7% C0046 30.8% 35.7% 42.2% C0050 26.7%34.5% 36.4% C0055 29.0% 34.9% 39.5% C0056 33.7% 38.9% 41.4% C0060 60.3%64.0% 68.0% C0086M 37.9% 48.1% 53.4% C0087M 51.6% 57.9% 61.5% C0088M40.1% 52.4% 56.1% C0089M 40.7% 46.1% 51.2% C0090M 42.5% 52.5% 55.8%C0091M 38.1% 39.8% 46.3% C0093M 44.8% 49.9% 53.5% C0094M 43.0% 52.8%57.5% C0095M 40.1% 46.6% 50.5% C0096M 43.0% 48.3% 55.0% C0099M 46.9%53.3% 56.0% C0100M 52.2% 58.2% 64.5% C0101M 50.5% 56.4% 59.0% C0102M52.3% 53.1% 56.6% C0104M 51.4% 54.1% 55.2% C0105M 55.7% 62.0% 68.8%C0106M 45.8% 55.6% 58.9% C0108M 54.6% 61.4% 68.7% C0114M 57.1% 63.2%66.7% C0115M 47.8% 57.8% 59.9% C0116M 53.9% 60.0% 62.9% C0118M 56.6%61.4% 62.4% C0119M 41.6% 55.5% 60.0% C0123M 51.9% 60.5% 62.9% C0124M47.7% 52.2% 58.7% C0125M 54.2% 59.7% 63.3% C0126M 50.7% 55.4% 67.3%C0128M 46.5% 54.4% 58.2% C0133M 47.8% 54.9% 58.5% C0134M 55.7% 60.5%61.9% F-C0134 37.4% 45.7% 53.1% C0135M 53.9% 55.1% 62.3% C0136M(P5)46.7% 55.2% 58.2% C0137M(P7) 42.4% 49.9% 61.2% C0142M 35.1% 39.4% 56.0%C0143M 45.3% 48.4% 57.8% C0148M 46.2% 52.0% 57.0% C0149M 48.5% 52.3%62.0% C0150M 47.3% 51.8% 61.4% C0151M 48.3% 51.7% 58.7% C0152M-4 ND NDND C0153M-3 ND ND ND Naloxone 39.87% 46.29% 50.91% Control Average *ND =Not Done.

A preliminary study similar to that immediately above was carried outusing Compounds 4, 9 and 10 and 100 nM of frozen-stored FITC-NLX ratherthan 10 nM FITC-NLX. The results of an average of two runs for thisstudy are shown below.

Compound 0.1 nM 1 nM 10 nM 100 nM 1 μM 4 18.8% 21.3% 17.9% 28.8% 42.9% 922.5% 24.8% 27.7% 35.3 49.6% 10 27.5% 27.3% 26.6% 27.3% 34.5% (+) NLX22.7% 22.8% 23.1% 22.8% 39.8%

Example 2 MOR Agonist Activity Using GTPγS Binding Assay

To assess the mu opiate receptor (MOR) agonist activity of positivecompounds from the FLNA screening, compounds were tested in a [³⁵S]GTPγSbinding assay using striatal membranes. A previous study has shown thatin striatal membranes, activation of MOR leads to an increase in[³⁵S]GTPγS binding to Gαo (Wang et al., 2005 Neuroscience 135:247-261).This assay measures a functional consequence of receptor occupancy atone of the earliest receptor-mediated events. The assay permits fortraditional pharmacological parameters of potency, efficacy andantagonist affinity, with the advantage that agonist measures are notsubjected to amplification or other modulation that may occur whenanalyzing parameters further downstream of the receptor.

Thus, striatal tissue was homogenized in 10 volumes of ice cold 25 mMHEPES buffer, pH 7.4, which contained 1 mM EGTA, 100 mM sucrose, 50μg/ml leupeptin, 0.04 mM PMSF, 2 μg/ml soybean trypsin inhibitor and0.2% 2-mercaptoethanol. The homogenates were centrifuged at 800×g for 5minutes and the supernatants were centrifuged at 49,000×g for 20minutes. The resulting pellets were suspended in 10 volume of reactionbuffer, which contained 25 mM HEPES, pH 7.5, 100 mM NaCl, 50 μg/mlleupeptin, 2 μg/ml soybean trypsin inhibitor, 0.04 mM PMSF and 0.02%2-mercaptomethanol.

The resultant striatal membrane preparation (200 μg) was admixed andmaintained (incubated) at 30° C. for 5 minutes in reaction buffer asabove that additionally contained 1 mM MgCl₂ and 0.5 nM [³⁵S]GTPγS (0.1μCi/assay, PerkinElmer Life and Analytical Sciences) in a total volumeof 250 μl and continued for 5 minutes in the absence or presence of0.1-10 μM of an assayed compound of interest. The reaction wasterminated by dilution with 750 μl of ice-cold reaction buffer thatcontained 20 mM MgCl₂ and 1 mM EGTA and immediate centrifugation at16,000×g for 5 minutes.

The resulting pellet was solubilized by sonicating for 10 seconds in 0.5ml of immunoprecipitation buffer containing 0.5% digitonin, 0.2% sodiumcholate and 0.5% NP-40. Normal rabbit serum (1 μl) was added to 1 ml oflysate and incubated at 25° C. for 30 minutes. Nonspecific immunecomplexes were removed by incubation with 25 μl of proteinA/G-conjugated agarose beads at 25° C. for 30 minutes followed bycentrifugation at 5,000×g at 4° C. for 5 minutes. The supernatant wasdivided and separately incubated at 25° C. for 30 minutes withantibodies raised against Gαo proteins (1:1,000 dilutions).

The immunocomplexes so formed were collected by incubation at 25° C. for30 minutes with 40 μl of agarose-conjugated protein A/G beads andcentrifugation at 5,000×g at 4° C. for 5 minutes. The pellet was washedand suspended in buffer containing 50 mM Tris-HCl, pH 8.0, and 1% NP-40.The radioactivity in the suspension was determined by liquidscintillation spectrometry. The specificity of MOR activation of[³⁵S]GTPγS binding to Gαo induced by a selective compound was defined byinclusion of 1 μM β-funaltrexamine (β-FNA; an alkylating derivative ofnaltrexone that is a selective MOR antagonist). DAMGO (1 or 10 μM) wasused as a positive control.

The results of this study are shown in the Tables below.

FLNA-Binding Compound MOR Agonist Activity

FLNA- Concentration of FLNA-Binding Compound as Agonist Binding 1 μM %DAMGO % DAMGO % DAMGO Compound 0.1 μM 1 μM +BFNA (0.1 μm) (1 μm) +BFNAA3333 170.7% 328.3% 65.9% 88.9% 101.0% 136.7% A0001 94.3% 181.7% 22.2%63.1% 78.9% 83.8% A0002 155.6% 199.4% 6.5% 104.1% 86.6% 24.5% A0003176.8% 276.0% 17.1% 118.3% 119.9% 64.5% A0004 97.4% 144.2% 86.0% 55.2%55.6% 130.9% A0005 179.7% 239.2% 23.5% 105.0% 89.6% 45.1% A0006 170.0%190.9% 18.2% 113.8% 82.9% 68.7% A0007 102.0% 221.9% 40.4% 68.3% 96.4%152.5% A0008 163.8% 235.0% 133.9% 109.6% 102.1% 505.3% A0009 70.2%126.4% 93.9% 39.8% 48.7% 142.9% A0010 277.2% 319.0% 190.3% 161.9% 119.5%365.3% A0011 236.3% 287.5% 47.0% 158.2% 124.9% 177.4% A0012 149.3%185.7% 122.4% 99.9% 80.7% 461.9% A0013 102.1% 164.8% 86.1% 57.8% 63.6%131.1% A0014 147.0% 174.9% 140.8% 83.2% 67.5% 214.3% A0015 110.9% 150.1%62.5% 64.8% 56.2% 120.0% A0017 161.9% 246.0% 65.2% 96.9% 100.4% 187.9%A0020 168.6% 217.4% 67.4% 100.9% 88.7% 194.2% A0021 133.3% 275.3% 12.1%79.8% 112.4% 34.9% A0022 154.1% 216.0% 28.0% 90.0% 80.9% 53.7% A002558.6% 138.7% 52.2% 33.2% 54.5% 198.5% A0026 140.7% 179.8% 120.8% 79.7%70.7% 459.3% A0028 143.6% 187.7% 116.7% 81.3% 73.8% 443.7% A0029 173.8%206.5% 22.3% 98.4% 81.2% 84.8% A0030 133.4% 287.8% 165.2% 75.5% 113.2%628.1% A0031 178.2% 297.0% 150.9% 100.9% 116.8% 573.8% A0032-1 187.4%324.5% 224.5% 95.5% 117.6% 303.8% A0032 226.9% 257.8% 133.0% 115.6%93.4% 180.0% A0033 155.8% 254.6% 118.2% 79.4% 92.2% 159.9% A0035 120.6%158.8% 88.6% 61.5% 57.5% 119.9% A0036 144.1% 167.5% 63.2% 73.4% 60.7%85.5% A0037 177.9% 236.2% 104.6% 90.7% 85.6% 141.5% A0038 176.7% 234.5%107.0% 90.1% 85.0% 144.8% A0039 267.8% 339.6% 173.5% 136.5% 123.0%234.8% A0040 46.1% 149.0% 16.7% 23.5% 54.0% 22.6% A0041 212.7% 283.6%50.6% 108.4% 102.8% 68.5% A0042 147.5% 233.1% 89.5% 75.2% 84.5% 121.1%A0043 183.3% 223.8% 89.1% 93.4% 81.1% 120.6% A0044 176.2% 209.1% 134.7%89.8% 75.8% 182.3% A0045 143.9% 274.2% 99.2% 73.3% 99.3% 134.2% A0046257.5% 354.1% 140.0% 131.2% 128.3% 189.4% A0047 233.0% 255.0% 116.5%118.8% 92.4% 157.6% A0048 233.7% 302.9% 167.2% 119.1% 109.7% 226.3%A0049 232.3% 370.3% 107.1% 118.4% 134.2% 144.9% A0050 151.0% 189.3%81.0% 77.0% 68.6% 109.6% A0051 290.4% 386.6% 211.6% 148.0% 140.1% 286.3%A0053 78.5% 118.2% 15.1% 46.5% 47.5% 46.2% A0054 74.9% 159.2% 114.1%44.4% 63.9% 348.9% A0055 89.8% 195.2% 33.5% 53.2% 78.4% 102.4% A0056115.6% 129.6% 17.4% 74.1% 56.2% 43.6% A0057 124.2% 192.1% 44.8% 79.6%83.3% 112.3% A0058 70.7% 244.3% 59.9% 45.3% 106.0% 150.1% A0059 99.2%129.9% 85.7% 63.5% 56.4% 214.8% A0060 99.7% 158.2% 14.3% 63.9% 68.6%35.8% A0061 110.3% 197.1% 10.7% 70.7% 85.5% 26.8% A0062 ND ND ND ND NDND A0063 122.8% 245.8%   310% 78.7% 106.6% 77.7% A0064 219.2% 262.9%43.7% 127.4% 119.7% 126.7% A0065 197.6% 266.8% 44.9% 126.6% 115.7%112.5% A0066 151.9% 195.6% 59.2% 88.3% 89.0% 171.6% A0067 170.8% 254.4%33.9% 99.2% 115.8% 98.3% A0068 73.9% 110.4% 98.1% 36.8% 35.2% 182.0%A0069 122.7% 244.2% 29.5% 71.3% 111.2% 85.5% A0070 128.6% 195.3% 80.3%74.7% 88.9% 232.8% A0071 225.7% 310.9% 239.4% 128.2% 122.9% 1088.2%A0072 254.3% 305.1% 171.8% 126.8% 97.2% 318.7% A0073 201.7% 325.7%185.8% 100.5% 103.7% 344.7% A0074 ND ND ND ND ND ND A0075 ND ND ND ND NDND A0076 79.8% 172.6% 41.2% 46.4% 78.6% 119.4% A0077 300.1% 334.7%103.5% 170.5% 132.3% 470.5% A0078 250.5% 289.9% 147.8% 124.9% 92.3%274.2%

Series B FLNA-Binding Compound MOR Agonist Activity

FLNA- Concentration of FLNA-Binding Compound as Agonist Binding 1 μM %DAMGO % DAMGO % DAMGO Compound 0.1 μM 1 μM +BFNA (0.1 μM) (1 μM) +BFNA5009 128.5% 270.4% 87.5% 66.9% 83.2% 181.5% B0001 128.2% 202.3% 28.0%77.4% 74.9% 43.1% B0002 165.7% 219.0% 101.4% 100.0% 81.1% 156.0% B0003103.0% 131.1% 18.6% 59.9% 47.4% 29.0% B0004 170.3% 231.7% 72.0% 102.8%85.8% 110.8% B0005 89.2% 110.4% 45.1% 50.5% 42.6% 68.6% B0006 77.0%131.3% 18.6% 44.8% 47.5% 29.0% B0007 168.3% 223.3% 64.5% 95.3% 86.1%98.2% B0008 148.3% 264.1% 46.0% 84.0% 101.9% 70.0% B0009 144.4% 219.9%119.4% 81.8% 84.8% 181.7% B0010 132.9% 184.4% 152.0% 75.3% 71.1% 231.4%B0011 158.6% 212.6% 78.0% 95.7% 78.7% 120.0% B0012 167.4% 212.0% 145.1%97.8% 79.4% 278.5% B0013 51.4% 154.1% 34.4% 29.1% 59.4% 52.4% B0014166.6% 250.5% 44.3% 98.5% 93.7% 67.1% B0016 167.7% 213.6% 72.2% 99.2%79.9% 109.4% B0017 99.6% 122.0% 49.6% 58.9% 45.6% 75.2% B0018 118.8%143.0% 45.6% 70.3% 53.5% 69.1% B0019 101.0% 256.5% 81.4% 59.7% 96.0%123.3% B0020 51.6% 181.6% 24.9% 30.1% 68.0% 47.8% B0021 126.9% 256.4%42.9% 75.9% 104.7% 123.6% B0022 131.9% 182.7% 45.8% 78.9% 74.6% 132.0%B0023 166.1% 245.3% 28.4% 99.4% 100.1% 81.8% B0024 155.8% 285.9% 20.2%93.2% 116.7% 58.2% B0025 159.6% 234.6% 137.7% 96.3% 86.8% 211.8% B0026152.0% 233.3% 28.8% 88.8% 87.4% 55.3% B0027 140.9% 266.9% 21.6% 82.3%100.0% 41.5% B0028 199.1% 357.7% 55.0% 103.5% 131.0% 125.3% B0029 171.9%210.3% 17.6% 89.4% 77.0% 40.1% B0030 107.2% 276.1% 90.1% 62.6% 103.4%172.9% B0031 210.8% 272.0% 28.8% 109.6% 99.6% 65.6% B0032 221.1% 297.7%15.6% 115.0% 109.0% 35.5% B0033 149.3% 188.9% 41.9% 77.6% 69.2% 95.4%B0034 122.5% 235.2% 41.8% 71.6% 88.1% 80.2% B0035 188.0% 248.7% 74.2%109.8% 93.2% 142.4% B0036 61.4% 120.6% 65.1% 39.2% 52.1% 199.7% B0037119.8% 186.0% 106.2% 76.5% 80.4% 325.8% B0038 147.5% 205.3% 117.1% 94.2%88.7% 359.2% B0039 171.8% 290.5% 78.3% 100.4% 108.8% 150.3% B0040 146.0%243.3% 55.3% 93.2% 105.1% 169.6% B0041 61.6% 109.3% 41.9% 39.3% 47.2%128.5% B0042 69.9% 107.5% 43.1% 39.6% 42.3% 163.9% B0043 74.8% 248.1%166.4% 42.4% 97.6% 632.7% B0044 87.3% 170.0% 134.6% 49.4% 66.9% 511.8%B0045 129.3% 193.1% 83.8% 82.6% 83.4% 257.1% B0046 99.9% 141.9% 90.5%63.8% 61.3% 277.6% B0047 187.8% 235.6% 68.4% 106.3% 92.6% 260.1% B0048185.1% 223.4% 78.5% 104.8% 87.8% 298.5% B0049 181.6% 364.0% 133.2%102.8% 143.1% 506.5% B0050 98.2% 211.0% 48.8% 58.1% 96.4% 294.0% B0051115.6% 167.9% 43.8% 68.4% 76.7% 263.9% B0052 98.2% 151.7% 40.9% 58.1%69.3% 246.4% B0053 160.2% 299.8% 134.3% 94.8% 137.0% 809.0% B0054 157.8%186.7% 111.0% 93.4% 85.3% 668.7% B0055 162.1% 338.5% 117.5% 91.8% 133.1%446.8% B0056 174.7% 288.8% 41.8% 98.9% 113.6% 158.9%

Series C-1 FLNA-Binding Compound MOR Agonist Activity

FLNA- Concentration of FLNA-Binding Compound as Agonist Binding 1 μM %DAMGO % DAMGO % DAMGO Compound 0.1 μM 1 μM +BFNA (0.1 μM) (1 μM) +BFNA7866 152.3% 308.2% 62.4% 79.3% 94.8% 129.5% C0001 129.3% 184.3% 33.9%75.2% 66.6% 52.9% C0002 88.4% 93.8% 3.9% 51.4% 33.9% 6.1% C0003 162.3%215.9% 107.7% 91.9% 83.3% 163.9% C0004 122.0% 228.4% 65.8% 72.1% 85.4%99.7% C0005 180.4% 227.2% 166.4% 105.4% 85.1% 319.4% C0006 121.5% 204.0%4.6% 70.6% 73.8% 7.2% C0007 79.1% 195.0% 10.9% 46.0% 70.5% 17.0% C000871.2% 201.6% 2.8% 41.4% 72.9% 4.4% C0009 146.3% 256.2% 26.4% 85.1% 92.6%41.2% C0010 136.5% 307.0% 89.1% 80.7% 114.9% 135.0% C0011 217.0% 305.0%19.0% 126.8% 114.3% 36.5% C0012 96.8% 224.8% 184.4% 54.8% 86.7% 280.7%C0013 156.6% 301.2% 39.6% 91.0% 108.9% 61.8% C0014 144.9% 153.5% 76.3%82.0% 59.2% 116.1% C0015 138.7% 204.7% 126.8% 78.5% 78.9% 193.0% C0016172.7% 230.5% 96.7% 100.4% 83.3% 150.9% C0017 153.8% 284.5% 94.1% 87.1%109.7% 143.2% C0018 195.5% 247.7% 106.5% 110.7% 95.5% 162.1% C0019104.4% 176.6% 52.8% 59.1% 68.1% 80.4% C0021 159.7% 192.0% 90.7% 94.5%87.8% 546.4% C0022 194.3% 328.7% 13.4% 113.5% 123.2% 25.7% C0023 153.2%233.7% 23.2% 89.5% 87.6% 44.5% C0024 178.4% 229.6% 59.3% 92.8% 84.1%135.1% C0025 235.7% 320.7% 80.2% 122.6% 117.5% 182.7% C0028 93.9% 132.4%78.4% 55.6% 60.5% 472.3% C0029 175.4% 308.8% 16.6% 91.2% 113.1% 37.8%C0030 150.3% 226.8% 95.0% 96.0% 98.0% 291.4% C0032 145.4% 202.0% 80.9%92.8% 87.3% 248.2% C0033 134.5% 186.4% 76.6% 85.9% 80.6% 235.0% C0034103.6% 167.9% 80.1% 61.3% 76.7% 482.5% C0041 186.1% 244.4% 95.5% 110.1%111.7% 575.3% C0042 167.1% 260.9% 110.6% 98.9% 119.2% 666.3% C0047142.2% 206.1% 80.1% 98.1% 88.5% 182.0% C0048 209.1% 245.3% 89.9% 144.2%105.3% 204.3% C0049 106.6% 210.0% 81.0% 73.5% 90.1% 184.1% C0051 94.4%170.4% 55.9% 65.1% 73.1% 127.0% C0052 108.4% 162.8% 42.7% 74.8% 69.9%97.0% C0053 104.0% 157.2% 93.1% 71.7% 67.5% 211.6% C0054 68.2% 127.0%43.5% 47.0% 54.5% 98.9% C0057  ND* ND ND ND ND ND C0061 ND ND ND ND NDND C0062 127.8% 310.5% 59.8% 81.9% 134.7% 149.9% C0064 213.8% 349.6%38.1% 124.2% 159.1% 110.4% C0065 198.3% 279.5% 47.7% 127.0% 121.3%119.5% C0067 142.7% 179.0% 33.5% 82.9% 81.5% 97.1% C0068 107.2% 263.1%165.9% 53.4% 83.8% 307.8% C0069 ND ND ND ND ND ND C0070 165.6% 210.8%114.2% 96.2% 95.9% 331.0% C0071 276.3% 355.3% 177.1% 160.5% 161.7%513.3% C0072 172.7% 259.1% 67.1% 100.3% 117.9% 194.5% C0073 ND ND ND NDND ND C0077 192.7% 265.4% 136.7% 109.5% 104.9% 621.4% C0078 138.1%236.6% 170.7% 82.4% 106.4% 359.4% C0080M 187.9% 205.4% 167.1% 112.1%92.4% 351.8% C0082M 228.1% 338.4% 97.6% 113.7% 107.8% 181.1% C0084M163.1% 255.5% 133.2% 97.3% 114.9% 280.4% C0085M 211.6% 246.2% 43.7%105.5% 78.4% 112.6% C0138M 126.9% 183.9% 51.5% 86.3% 90.9% 131.0% C0139M156.1% 206.6% 51.0% 106.2% 102.2% 129.8% C0140M 126.1% 215.4% 83.0%85.8% 106.5% 211.2% C0141M 161.5% 213.9% 47.9% 109.9% 105.8% 121.9%C0143M 81.0 193.3 86.5 47.1% 59.3% 94.7% C0144M 186.3 295.9 125.9 108.3%90.8% 137.9% C0145M 193.0 289.2 87.0 112.2% 88.7% 95.3% C0146M ND ND NDND ND ND C0147M A2 ND ND ND ND ND ND C0148M A2 181.3 360.6 87.6 105.4%110.6% 95.9% C0149M 209.8 406.7 93.4 122.0% 124.8% 102.3% C0150M 167.1423.1 93.4 97.2% 129.8% 173.2% C0151M 346.8 397.6 212.8 201.6% 122.0%233.1% C0152M ND ND ND ND ND ND DAMGO 168.5% 266.1% 53.2% ND ND NDAverage *ND =Not Done.

Series C-2 FLNA-Binding Compound MOR Agonist Activity

FLNA- Concentration of FLNA-Binding Compound as Agonist Binding 1 μM %DAMGO % DAMGO % DAMGO Compound 0.1 μM 1 μM +BFNA (0.1 μM) (1 μM) +BFNAC0011 217.0% 305.0% 19.0% 126.8% 114.3% 36.5% C0026 207.2% 288.4% 21.2%107.7% 105.6% 48.3% C0027 233.2% 313.9% 72.2% 121.3% 115.0% 164.5%S-C0027 156.2% 286.8% 56.2% 74.2% 84.4% 98.1% C0034-3  ND* ND ND ND NDND C0037-2 ND ND ND ND ND ND C0040 145.8% 308.3% 90.4% 93.1% 133.2%277.3% C0043 175.4% 242.6% 83.3% 103.8% 110.9% 501.8% C0044 173.7%280.1% 59.1% 102.8% 128.0% 356.0% C0045 149.2% 238.8% 105.3% 88.3%109.1% 634.3% C0046 286.2% 492.9% 156.8% 197.4% 211.5% 356.4% C0050110.3% 127.6% 59.0% 76.1% 54.8% 134.1% C0055 ND ND ND ND ND ND C005698.6% 193.4% 86.3% 68.0% 83.0% 196.1% C0060 166.5% 218.9% 143.9% 114.8%93.9% 327.0% C0086M 206.8% 265.3% 152.3% 117.5% 104.9% 692.3% C0087M262.8% 329.6% 142.5% 138.9% 132.8% 293.8% C0088M 276.3% 355.3% 177.1%160.5% 161.7% 513.3% C0089M 234.5% 295.3% 81.9% 136.3% 134.4% 237.4%C0090M 237.0% 341.0% 41.0% 137.7% 155.2% 118.8% C0091M 207.9% 274.4%80.8% 118.1% 108.5% 367.3% C0093M 140.0% 211.8% 44.0% 81.3% 96.4% 127.5%C0094M 172.5% 263.5% 115.3% 100.2% 119.9% 334.2% C0095M 189.1% 224.6%107.7% 107.4% 88.8% 489.5% C0096M 186.4% 328.9% 127.1% 105.9% 130.0%577.7% C0099M 157.2% 195.7% 114.7% 93.8% 88.0% 241.5% C0100M 173.6%245.9% 195.6% 103.6% 110.6% 411.8% C0101M 138.2% 274.3% 174.8% 82.5%123.4% 368.0% C0102M 131.8% 272.0% 150.4% 78.6% 122.4% 316.6% C0104M188.2% 238.9% 143.8% 99.5% 96.3% 296.5% C0105M 198.1% 220.3% 73.1%104.7% 88.8% 150.7% C0106M 171.8% 240.7% 117.2% 102.5% 108.3% 246.7%C0108M 205.6% 258.5% 76.9% 108.7% 104.1% 158.6% C0114M 114.0% 144.3%35.9% 77.6% 71.4% 91.3% C0115M 177.2% 226.8% 118.4% 105.7% 102.0% 249.3%C0116M 258.4% 302.8% 152.0% 136.6% 122.0% 313.4% C0118M 166.2% 261.5%79.2% 87.8% 105.4% 163.3% C0119M 105.7% 167.8% 35.1% 71.9% 83.0% 89.3%C0124M 252.0% 305.1% 61.4% 133.2% 122.9% 126.6% C0125M 168.6% 195.2%159.7% 89.1% 78.6% 329.3% C0126M 181.8% 265.3% 108.5% 108.5% 119.3%228.4% C0128M 197.8% 286.0% 63.9% 104.5% 115.2% 131.8% C0133M 139.4%214.8% 72.4% 83.2% 96.6% 152.4% C0134M 158.5% 207.3% 46.6% 94.6% 93.3%98.1% F-00134 290.6% 378.9% 66.6% 138.1% 111.4% 116.2% C0135M 161.3%310.1% 113.3% 85.3% 124.9% 233.6% C0136M (P5) 176.8% 237.3% 74.5% 93.4%95.6% 153.6% C0137M (P7) 180.8% 193.8% 55.8% 95.6% 78.1% 115.1% C0142M143.7% 192.5% 98.7% 97.8% 95.2% 251.1% C0143M 81.0% 193.3% 86.5% 47.1%59.3% 94.7 C0144M-21 86.3% 295.9% 125.9% 108.3% 90.8% 137.9% C0145M-3193.0% 289.2% 87.0% 112.2% 88.7% 95.3% C0149M-22 09.8% 406.7% 93.4%122.0% 124.8% 102.3% C0150M-21 67.1% 423.1% 158.1% 97.2% 129.8% 173.2%C0151M-23 46.8% 397.6% 212.8% 201.6% 122.0% 233.1% C0152M-2 ND ND ND NDND ND C0153M-3 ND ND ND ND ND ND DAMGO 168.5% 266.1% 53.2% ND ND NDAverage *ND = Not Done.

A preliminary study similar to that immediately above was carried outusing Compounds 4, 9 and 10 and resynthesized Compound C0134M and DAMGO.The results of an average of two runs for this study are shown below.

Concentration of FLNA-Binding Compound as Agonist Compound 0.1 μM 1 μM 1μM + βNFA 4 133.9% 165.2% 49.5% 9 156.6% 197.2% 56.6% 10 163.1% 191.8%60.4% C0134M 150.7% 224.0% 53.2% DAMGO 144.7% 233.4% 56.8%

The above results indicate that Compounds 9 and 10 not only bind well toFLNA, but are also MOR agonists, whereas Compound 4 bound well to FLNA,but was not as potent a MOR agonist as were the other two compounds. Thenewly synthesized Compound C0134M exhibited similar MOR agonist activityto that shown previously.

Materials and Methods

An in vitro study was conducted under the direction of Hoau-Yan Wang,Ph.D. by the Dept. of Physiology, Pharmacology & Neuroscience, CUNYMedical School, 138th Street and Convent Avenue, New York, N.Y. 10031,to assess the top two filamin A (FLNA)-binding compounds, C0105 andC0114 for the ability to block amyloid beta₄₂ (Aβ₄₂)-induced FLNA-α7nicotinic acetylcholine receptor (α7nAChR) association and tauphosphorylation, indicating the potential to treat Alzheimer's disease.

Animals

Adult Sprague Dawley rats (2 months old) were used for organotypicfrontocortical slice cultures. Rats were maintained on a 12-hourlight/dark cycle with food and water. All animal procedures comply withthe National Institutes of Health Guide for Care Use of LaboratoryAnimals and were approved by the City College of New York Animal Careand Use Committee.

Organotypic Frontocortical Slice Cultures

Rat brain slice organotypic culture methods were modified from thosepublished previously. [Adamchik et al., Brain Res Brain Res Protoc5:153-158 (2000).] FCX slices (200 μM thickness) were transferred tosterile, porous Millicell-CM inserts (0.4 μm). Each culture insert unitcontained two brain slices and was placed into individual wells of a12-well culture tray in 2 ml medium: 50% MEM with Earl's salts, 2 mML-glutamine, 25% Earl's balanced salt solution, 6.5 g/l D-glucose, 20%fetal bovine serum (FBS), 5% horse serum, 25 mM HEPES buffer, pH 7.2,and 50 mg/ml streptomycin and 50 mg/ml penicillin. Cultures were kept inan incubator for 2 days at 36° C. in 5% CO₂ to minimize the impact ofinjury from slice preparation.

On the day of experiment, medium was removed, the brain slices rinsedand incubated in 0.1% FBS-containing medium for 4 hr at 36° C. in 5%CO₂. Brain slices were then cultured with 100 nM Aβ₄₂ and/or 0.1, 1 nMcompound C0105 or compound C0114 in fresh 0.1% FBS-containing medium for16 hours. Brain slices (6 slices for each experiment) were washed withice-cold Krebs-Ringer and used to assess α7nAChR-FLNA complex level andphosphorylated tau (pS²⁰²-, pT²³¹- and pT¹⁸¹-tau). Brain slices werealso used to determine the α7nAChR and NMDAR activity by the level ofcalcium influx through each of these two channels and the level of celldeath using voltage-gated calcium channel mediated calcium influx.

For immunohistochemistry, additional slices were removed and fixed in 4%paraformaldehyde in PBS at 4° C. The effect of C0105 and C0114 onintraneuronal Aβ₄₂ accumulation was determined by the Aβ₄₂immunostaining level.

Brain Synaptosome Preparation

Brain synaptosomes (P2 fraction) were prepared from FCX slice cultures.Following methods described previously, [Wang et al., J Biol Chem278:31547-31553 (2003)] FCX was solubilized immediately after removalfrom cultures to obtain synaptosomes. The synaptosomes were washed twiceand suspended in 2 ml of ice-cold Kreb's-Ringer (K-R): 25 mM HEPES, pH7.4; 118 mM NaCl, 4.8 mM KCl, 25 mM NaHCO₃, 1.3 mM CaCl₂, 1.2 mM MgSO₄,1.2 mM KH₂PO₄, 10 mM glucose, 100 μM ascorbic acid, mixture of proteaseand protein phosphatase inhibitors (Roche Diagnostics) that had beenaerated for 10 minutes with 95% O₂/5% CO₂. The protein concentration wasdetermined using the Bradford method (Bio-Rad).

In Vitro Studies Using Organotypic FCX Tissues

To assess the effect of compounds C0105 and C0114 on Aβ₄₂-inducedα7nAChR-FLNA interaction and tau phosphorylation (pS²⁰²-, pT²³¹- andpT¹⁸¹-tau) levels, rat frontal cortical slice culture system was used.Rat brain FCX were chopped coronally into 200 μm slices using a Mcllwainchopper (Brinkman Instruments) and suspended in 10 ml of ice-coldoxygenated K-R.

The rat brain slice organotypic culture was performed as describedpreviously. [Wang et al., Biol Psychiatry 67, 522-530 (2010).] Rat FCXslices were transferred to sterile, porous 0.4 μm Millicell-CM insert, 2slices per insert per well containing 2 ml medium: 50% MEM with Earl'ssalts, 2 mM L-glutamine, 25% Earl's balanced salt solution, 6.5 g/lD-glucose, 20% fetal bovine serum (FBS), 5% horse serum, 25 mM HEPESbuffer, pH 7.2, and 50 mg/ml streptomycin and 50 mg/ml penicillin.Cultures were kept in an incubator for 2 days at 36° C. in 5% CO₂.

On the day of study, medium was removed, the brain slices rinsed andincubated in 0.1% FBS-containing medium for 4 hours at 36° C. in 5% CO₂.Brain slices were then cultured with 0.1 μM Aβ₄₂ together with 0.1, 1 or10 nM Compound C0105 or 1 or 10 nM C0114 in fresh 0.1% FBS-containingmedium for 16 hours. The brain slices were then removed and washed withice-cold PBS three times and either processed for functional assaysdescribed below or fixed in ice-cold 4% paraformaldehyde PBS at 4° C.for determination of intraneuronal Aβ₄₂ aggregate and NFT levels byimmunohistochemical method.

1) FLNA Association with α7nAChR and Other Receptors

The level of FLNA-associated α7nAChRs was determined using aco-immunoprecipitation/Western blotting method as described previously.[Wang et al., Biol Psychiatry 67:522-530 (2010); Wang et al., PLoS One3:e1554 (2008); Wang et al., J Neurosci 35:10961-10973 (2009)] Briefly,brain slice extract (200 μg) was incubated with 1 μg anti-FLNAimmobilized on protein A agarose beads at 4° C. overnight (about 18hours) with constant end-over-end rotation. The anti-FLNAimmunocomplexes were obtained by centrifugation, and then washed anddissociated using antigen elution buffer. Following neutralization with1.5M Tris, pH 8.8, the resultant FLNA-associated protein complexes weresolubilized by boiling for 5 minutes in SDS-containing samplepreparation buffer. The levels of FLNA-associated α7nAChR, TLR4, IR andMOR were assessed by Western blotting using specific antibodies directedagainst the respective proteins and the blot stripped and re-probed forFLNA for immunoprecipitation/loading control.

2) Tau Phosphorylation

Using an established method, [Wang et al., J Biol Chem 278:31547-31553(2003); Wang et al., Biol Psychiatry 67:522-530 (2010)] tau proteinswere immunoprecipitated with immobilized anti-tau (SC-65865), which doesnot discriminate between phosphorylation states. The levels ofphosphorylated tau (pSer202tau, pThr231tau and pThr181tau) as well astotal tau precipitated (loading controls) are assessed by Westernblotting using specific antibodies directed against each of thephosphoepitopes and the anti-tau, respectively.

3) Functional Assessment of α7nAChR and NMDAR

The effect of Compounds C0105 and C0114 on α7nAChR and NMDAR functionwas assessed in organotypic FCX slice cultures treated with vehicle, 0.1μM Aβ₄₂ or 0.1 μM Aβ₄₂+0.1-10 nM C0105 or 1-10 nM C0114. Synaptosomesprepared from rat FCX slices (6 slices/assay) were washed twice inice-cold K-R, centrifuged and re-suspended in 0.5 ml K-R.

NMDAR and α7nAChR mediated ⁴⁵Ca²⁺ influx was measured as describedpreviously. [Wang et al., Biol Psychiatry 67:522-530 (2010).]Synaptosomes (50 μg) were incubated at 37° C. for 5 minutes inoxygenated 0.3 mM Mg²⁺ K-R containing 5 μM ⁴⁵Ca²⁺ (10 Ci/mmol,PerkinElmer) followed by incubation with vehicle, 0.1-10 μM NMDA/1 μMglycine or 0.1-10 μM PNU282987 (a potent and selective agonist for theα7 subtype of neural nicotinic acetylcholine receptors) for 5 minutes.The reaction was terminated by 1 ml ice-cold 0.5 mM EGTA-containingCa²⁺-free K-R and centrifugation. After two washes, synaptosomal ⁴⁵Ca²⁺contents were assessed using scintillation spectrometry.

The background ⁴⁵Ca²⁺ was estimated using hypotonically lysedsynaptosomes. The absolute Ca²⁺ influx was calculated by subtractingbackground ⁴⁵Ca²⁺ count. The percent increase in Ca²⁺ influx wascalculated as % [(drug-treated-vehicle)/vehicle].

4) Cell Death Measured by K⁺-Evoked Ca⁺² Influx

Because the level of voltage-gated Ca²⁺ channel activity is indicativeof the integrity of the cells, the effect of compounds C0105 and C0114on Aβ₄₂-induced cell death was assessed in organotypic FCX slicecultures treated with vehicle, 0.1 μM Aβ₄₂ or 0.1 μM Aβ₄₂+0.1-10 nMcompound C0105 or 1-10 nM compound C0114 using K⁺-depolarizationmediated Ca²⁺ influx. Synaptosomes prepared from rat FCX slices (6slices/assay) were washed twice in ice-cold K-R, centrifuged andre-suspended in 0.5 ml K-R. The level of voltage-gated Ca²⁺ channelmediated ⁴⁵Ca²⁺ influx was measured as described previously. [Wang etal., Biol Psychiatry 67:522-530 (2010).]

Synaptosomes (50 μg) were incubated at 37° C. for 5 minutes inoxygenated 0.3 mM Mg²⁺ K-R containing 5 μM ⁴⁵Ca²⁺ (10 Ci/mmol,PerkinElmer) followed by incubation with vehicle or 65 mM K⁺ (made withisomolar replacement of Na⁺) for 1 minute. The reaction was terminatedby 1 ml ice-cold 0.5 mM EGTA-containing Ca²⁺-free K-R andcentrifugation. After two washes, synaptosomal ⁴⁵Ca²⁺ contents wereassessed using scintillation spectrometry. The background ⁴⁵Ca²⁺ wasestimated using hypotonically lysed synaptosomes. The absolute Ca²⁺influx was calculated by subtracting background ⁴⁵Ca²⁺ count. Thepercent increase in Ca²⁺ influx was calculated as %[(drug-treated-vehicle)/vehicle].

5) Measuring Levels of Signaling Molecules Associated with NMDAR or IRafter Receptor Stimulation

NMDAR signaling and their interaction with synaptic anchoring protein,PSD-95, were compared in brain slices from organotypic culture FCXtreated with vehicle, 0.1 μM Aβ₄₂ and 0.1 μM Aβ₄₂+0.1-10 nM of compoundC0105 or 1 and 10 nM of compound C0114 for 16 hours. NMDAR activationand signaling was initiated by incubation of 6 slices with either 0.3 mMMg²⁺ containing KR (LMKR; basal) or LMKR containing 10 μM NMDA and 1 μMglycine at 37° C. for 30 minutes.

The incubation mixture was aerated with 95% O₂/5% CO₂ every 10 minutesfor 1 minute during the stimulation. Ligand stimulation was terminatedby the addition of 1 ml of ice-cold Ca²⁺-free K-R containing mixture ofprotein phosphatase inhibitors, 0.5 mM EGTA and 0.1 mM EDTA. Brainslices were harvested by a brief centrifugation and were homogenized in0.25 ml of ice-cold immunoprecipitation buffer. The homogenates werecentrifuged at 1000×g for 5 minutes (4° C.) and the supernatant(post-mitochondrial fraction) was sonicated for 10 seconds on ice.

The proteins were solubilized in 0.5% digitonin, 0.2% sodium cholate and0.5% NP-40 for 60 minutes at 4° C. with end-over-end rotation. Theresultant lysates were cleared by centrifugation at 50,000×g for 5minutes and diluted with 0.75 ml of immunoprecipitation buffer. Proteinconcentrations were measured by Bradford method (Bio-Rad).

To determine the association of NMDARs with PSD-95, as well as NMDARsignaling, the levels of NMDAR subunits, PSD-95 and NMDAR-associatedsignaling molecules were measured in anti-NR1 immunoprecipitates. Inthese studies, brain slice lysates (100 μg) were immunoprecipitatedovernight at 4° C. with 2 μg of immobilized anti-NR1 onto covalentlyconjugated protein A-agarose beads (Pierce-ENDOGEN). Anti-NR1immunoprecipitates were incubated with 75 μl antigen elution buffer(Pierce-ENDOGEN) and 2% SDS for 2 minutes on ice, centrifuged to removeantibody-protein A-agarose complexes and neutralized immediately with 10μl 1.5 M Tris buffer, pH 8.8 followed by addition of 65 μl 2× PAGEsample buffer and boiled for 5 minutes.

Seventy-five μl of the obtained eluates (50%) were size fractionated on7.5% SDS-PAGE. Proteins were transferred to nitrocellulose membrane andthe levels of various NMDA receptor subunits, PSD-95, signaling proteinswere measured using Western blotting with antibodies for PSD-95, nNOS,phospholipase C-γ1, γPKC, pY⁴⁰²PyK2, pY⁴¹⁶Src or phosphotyrosine. Theblots were stripped and re-probed with anti-NR1 to assess theimmunoprecipitation efficiency and loading.

IR activation and signaling was initiated by incubation of 6 slices thatwere further chopped horizontally into 100 μm (100 μm×200 μm×3 mm) witheither KR (basal) or KR containing 1 nM insulin at 37° C. for 30minutes. The incubation mixture was aerated with 95% O₂/5% CO₂ every 10minutes for 1 minute during the stimulation. Ligand stimulation wasterminated by the addition of 1 ml of ice-cold Ca²⁺-free K-R containingmixture of protein phosphatase inhibitors, 0.5 mM EGTA and 0.1 mM EDTA.Brain slices were harvested by a brief centrifugation and werehomogenized in 0.25 ml of ice-cold immunoprecipitation buffer. Thehomogenates were centrifuged at 1000×g for 5 minutes (4° C.) and thesupernatant (post-mitochondrial fraction) was sonicated for 10 secondson ice. The proteins were solubilized in 0.5% digitonin, 0.2% sodiumcholate and 0.5% NP-40 for 60 minutes at 4° C. with end-over-endrotation. The resultant lysates were then cleared by centrifugation at50,000×g for 5 minutes and diluted with 0.75 ml of immunoprecipitationbuffer. Protein concentrations were measured by Bradford method(Bio-Rad).

To determine the IR activation and signaling, the levels ofpY^(1150/1151)IRB and the level of IR signal transducer, IRS-1 weremeasured in anti-IV immunoprecipitates. In these experiments, brainslice lysates (100 μg) were immunoprecipitated overnight (about 18hours) at 4° C. with 2 μg of immobilized anti-IRβ onto covalentlyconjugated protein A-agarose beads (Pierce-ENDOGEN).

Anti-IRβ immunoprecipitates were incubated with 75 μl antigen elutionbuffer (Pierce-ENDOGEN) and 2% SDS for 2 min on ice, centrifuged toremove antibody-protein A-agarose complexes and neutralized immediatelywith 10 μl 1.5 M Tris buffer, pH8.8 followed by addition of 65 μl 2 XPAGE sample buffer and boiled for 5 minutes. Seventy-five μl of theobtained eluates (50%) were then size fractionated on 7.5% SDS-PAGE.Proteins were transferred to nitrocellulose membrane and the levels ofpY^(1150/1151)IRβ and IRS-1 proteins were measured using Westernblotting with antibodies for pY^(1150/1151)IRβ and IRS-1. The blots werestripped and re-probed with anti-IRβ to assess the immunoprecipitationefficiency and loading.

6) Immunohistochemical Studies

Quantitative immunohistochemistry on consecutive 5-μm sectionscontaining PFCX and entorhinal cortex/HP were used to determine thelevels of Aβ₄₂ aggregates/plaques and neurofibrillary pathology (NFT andpaired helical filament [PHF] immunoreactivity) using single labelingimmunohistochemistry as described previously. [Wang et al., BiolPsychiatry 67:522-530 (2010); D'Andrea et al., Histopathology 38:120-134(2001); Nagele et al., Neuroscience 110:199-211 (2002).] One section wasimmunostained with anti-NFT or -PHF. The next (consecutive) section(often containing the same neuron) was immunostained with anti-Aβ₄₂antibodies to measure relative levels of accumulated Aβ₄₂ peptide inneurons. The relative Aβ₄₂ accumulation rate/extent were compared amongdifferent cell types in sections from cultured FCX slices and icvAβ₄₂-infused mouse brains using a computer-assisted image analysis asdescribed previously [Wang et al. J Biol Chem 275:5626-5632 (2000)].

Brain slices were fixed at 4° C. in 0.15 M phosphate-buffered 10%formalin, pH 7.4 for 2 weeks, paraffin embedded, serially sectioned at 5μm, and processed for brightfield immunohistochemistry as describedpreviously [Wang et al., J Biol Chem 275:5626-5632 (2000)]. The Aβ₄₂immunoreactivity was absent when pre-absorbing anti-Aβ₄₂ with Aβ₄₂ butnot Aβ₄₂₋₁. Specimens were examined using a Nikon FXA microscope with aPrinceton Instruments CCD camera and recorded digitally.

Relative intensities of the NFT/PHF and Aβ₄₂ immunoreactivity weremeasured and compared among similar and different cell types using ImagePro Plus and Metamorph software as described previously [D'Andrea etal., Histopathology 38:120-134 (2001)]. The correlations between theamount of NFT/PHF immunoreactivity and Aβ₄₂-positive materialaccumulated within mature neurons were also determined.

In Vivo Studies

An in vivo study was conducted under the direction of Hoau-Yan Wang,Ph.D. by the Dept. of Physiology, Pharmacology & Neuroscience, CUNYMedical School, 138th Street and Convent Avenue, New York, N.Y. 10031,in an amyloid beta₄₂ (Aβ₄₂) infusion model of Alzheimer's disease forthe ability 1) to block Aβ₄₂-induced FLNA association with α7 nicotinicacetylcholine receptor (α7nAChR) and toll-like receptor 4 (TLR4), 2) tauphosphorylation, and 3) Aβ₄₂-α7nAChR association indicating thepotential to treat Alzheimer's disease.

ICV Aβ₄₂ Infusion Mouse Model

Mice

Eight-week-old male and female E129 mice (30-35 g), progeny of thebreeding pairs from Taconic (Germantown, N.Y.) were used in theintracerebroventricular (ICV) Aβ₄₂ study. Mice were maintained on a12-hour light/dark cycle with food and water. All animal procedurescomply with the National Institutes of Health Guide for Care Use ofLaboratory Animals and were approved by the City College of New YorkAnimal Care and Use Committee.

Intracerebroventricular Aβ₄₂ Administration and Compound Treatment

Mice anesthetized with 30 mg/kg sodium pentobarbital intraperitoneallywere placed in a mouse stereotaxic surgery apparatus as described byWang et al., Biol Psychiatry 67:522-530 (2010). Mice receiving 7-daycontinuous ICV Aβ₄₂ infusion were implanted with a minipump for mice(Alzet) that delivers 0.1 μl/hr through a surgical glue-secured cannulaplaced in the left ventricle at the following coordinates:[anterior-posterior from bregma, 3.0 mm; lateral, 1.0 mm; horizontal,3.0 mm]. The Aβ₄₂ (0.2 nmol/μl) was dissolved in 10% DMSO containing 50mM Tris, pH 9.0, to prevent aggregation. Each mouse received 4.8 nmolAβ₄₂ daily for 7 days. Control mice received 7-day ICV infusion ofvehicle.

To assess the effect of in vivo Compound C0105 on Aβ₄₂-elicited effects,mice received 10 mg/kg of Compound C0105 by intraperitoneal (i.p.)injection daily for 2 weeks starting on the day of surgery (day 1: 2hours after recovery from surgery, day 2-14 twice daily: between 10-11a.m. and 3-4 p.m.). Twenty-four hours after the last injection, FCX andhippocampus from one half brain was solubilized for assessment ofα7nAChR-FLNA complex level and phosphorylated tau (pS²⁰²-, pT²³¹- andpT¹⁸¹-tau) using published methods [Wang et al., Biol Psychiatry67:522-530 (2010)].

Whether the compounds has an effect on levels of Aβ₄₂-α7nAChR couplingwas assessed because dissociating Aβ₄₂ from α7nAChRs is beneficial inreducing AD pathologies. [Wang et al., Biol Psychiatry 67:522-530(2010); Wang et al., J Neurosci 35:10961-10973 (2009).] In addition,prefrontal cortex (PFCX) is used to determine the level of synapticactivity using α7nAChR and NMDAR activity as the guide. The other brainhalves were immersion-fixed in cold 0.15 M phosphate-buffered 10%formalin, pH 7.4, and processed for immunohistochemical determinationsof intraneuronal Aβ₄₂ aggregates/plaques and NFTs as well asmorphological integrity.

Brain Synaptosome Preparation

Brain synaptosomes (P2 fraction) were prepared from prefrontal cortexand hippocampus of treated mice sacrificed by rapid decapitation.Following methods described previously [Wang et al., J Biol Chem278:31547-31553 (2003)], tissue was solubilized immediately afterharvesting to obtain synaptosomes. The synaptosomes were washed twiceand suspended in 2 ml of ice-cold Kreb's-Ringer (K-R): 25 mM HEPES, pH7.4; 118 mM NaCl, 4.8 mM KCl, 25 mM NaHCO₃, 1.3 mM CaCl₂, 1.2 mM MgSO₄,1.2 mM KH₂PO₄, 10 mM glucose, 100 μM ascorbic acid, mixture of proteaseand protein phosphatase inhibitors (Roche Diagnostics) that had beenaerated for 10 minutes with 95% O₂/5% CO₂. The protein concentration wasdetermined using the Bradford method (Bio-Rad).

Ex Vivo Assessments of Tissues from Treated Mice

Using synaptosomes prepared from prefrontal cortex or hippocampi of micereceiving continuous ICV infusions of vehicle or Aβ₄₂ and twice dailyi.p. injections of Compound C0105 or vehicle, these studies assessed theeffect of Compound C0105 on Aβ₄₂-induced α7nAChR-FLNA interaction, tauphosphorylation (pS²⁰²-, pT²³¹- and pT¹⁸¹-tau) levels, Aβ₄₂-α7nAChRinteraction and signaling impairments.

1) α7nAChR-FLNA/TLR4 Interaction

The level of FLNA-associated α7nAChRs and TLR4s were determined using aco-immunoprecipitation/Western blotting method as described previously[Wang et al., Biol Psychiatry 67:522-530 (2010); Wang et al., J Neurosci35:10961-10973 (2009); and Wang et al., PLoS One 3:e1554 (2008)].Briefly, synaptosomal extracts (200 μg) prepared from prefrontal cortexor hippocampus from treated mice were incubated with 1 μg anti-FLNAimmobilized on protein A agarose beads at 4° C. overnight (about 18hours) with constant end-over-end rotation. The anti-FLNAimmunocomplexes were obtained by centrifugation, washed and dissociatedusing antigen elution buffer. Following neutralization with 1.5M Tris,pH 8.8, the resultant FLNA-associated protein complexes were solubilizedby boiling for 5 minutes in SDS-containing sample preparation buffer.The levels of FLNA-associated α7nAChRs and TLR4s were assessed byWestern blotting and the blot stripped and re-probed for FLNA forimmunoprecipitation/loading control.

To assess the effect of elevated Aβ₄₂ and Compound C0105 treatment onFLNA and α7nAChR expression, FLNA and α7nAChR levels were measured inthe tissue extract by Western blotting with β-actin as the loadingcontrol.

2) Tau Phosphorylation

Using established methods [Wang et al., Biol Psychiatry 67:522-530(2010); and Wang et al., J Biol Chem 278:31547-31553 (2003)], tauproteins were immunoprecipitated with immobilized anti-tau (SC-65865),which does not discriminate between phosphorylation states. The levelsof phosphorylated tau (pSer202tau, pThr231tau and pThr181tau) as well astotal tau precipitated (loading controls) were assessed by Westernblotting using specific antibodies directed against each of thephosphoepitopes and the anti-tau, respectively.

3) Aβ₄₂-α7nAChR Interaction

The level of Aβ₄₂-α7nAChR complexes were measured in synaptosomes fromprefrontal cortex and hippocampus of treated mice using an establishedmethod [Wang et al., Biol Psychiatry 67:522-530 (2010); and Wang et al.,J Biol Chem 278:31547-31553 (2003)]. Briefly, Aβ₄₂-α7nAChR complexeswere immunoprecipitated with immobilized anti-Aβ₄₂ and the α7nAChRcontents were measured by Western blotting. Anti-actin was added toimmunoprecipitation and the β-actin level in the immunoprecipitatesserved as immunoprecipitation/loading control.

4) Functional Assessment of α7nAChR and NMDAR

The effect of Compound C0105 on α7nAChR and NMDAR function was assessedin mice infused with Aβ₄₂ or vehicle. Synaptosomes prepared fromprefrontal cortex or hippocampus were washed twice in ice-cold K-R,centrifuged and re-suspended in 0.5 ml K-R.

NMDAR and α7nAChR mediated ⁴⁵Ca²⁺ influx was measured as describedpreviously [Wang et al., Biol Psychiatry 67:522-530 (2010)].Synaptosomes (50 μg) were incubated at 37° C. for 5 minutes inoxygenated 0.3 mM Mg²⁺ K-R containing 5 μM ⁴⁵Ca²⁺ (10 Ci/mmol,PerkinElmer) followed by incubation with vehicle, 0.1-10 μM NMDA/1 μMglycine or 0.1-10 μM PNU282987 for 5 minutes. The reaction wasterminated by admixture of 1 ml ice-cold 0.5 mM EGTA-containingCa²⁺-free K-R and centrifugation.

After two washes, synaptosomal ⁴⁵Ca²⁺ contents were assessed usingscintillation spectrometry. The background ⁴⁵Ca²⁺ was estimated usinghypotonically lysed synaptosomes. The absolute Ca²⁺ influx wascalculated by subtracting background ⁴⁵Ca²⁺ count. The percent increasein Ca²⁺ influx was calculated as % [(drug-treated-vehicle)/vehicle].

5) Cell Death Measured by K⁺-Evoked Ca+2 Influx

Because the level of voltage-gated Ca²+ channel activity is indicativeof the integrity of the cells, the effect of Compound C0105 onAβ₄₂-induced cell death was assessed in treated mice usingK⁺-depolarization mediated Ca²⁺ influx. Synaptosomes prepared fromprefrontal cortex were washed twice in ice-cold K-R, centrifuged andre-suspended in 0.5 ml K-R.

The level of voltage-gated Ca²⁺ channel mediated ⁴⁵Ca²⁺ influx wasmeasured as described previously [Wang et al., Biol Psychiatry67:522-530 (2010)]. Synaptosomes (50 μg) were incubated at 37° C. for 5minutes in oxygenated 0.3 mM Mg²⁺ K-R containing 5 μM ⁴⁵Ca²⁺ (10Ci/mmol, PerkinElmer) followed by incubation with vehicle or 65 mM K⁺(made with isomolar replacement of Na⁺) for 1 minute. The reaction wasterminated by admixture of 1 ml ice-cold 0.5 mM EGTA-containingCa²⁺-free K-R and centrifugation.

After two washes, synaptosomal ⁴⁵Ca²⁺ content was assessed usingscintillation spectrometry. The background ⁴⁵Ca²⁺ was estimated usinghypotonically lysed synaptosomes. The absolute Ca²⁺ influx wascalculated by subtracting background ⁴⁵Ca²⁺ count. The percent increasein Ca²⁺ influx was calculated as % [(drug-treated-vehicle)/vehicle].

6) Measuring Levels of Signaling Molecules Associated with NMDAR or IRafter Receptor Stimulation

NMDAR signaling and their interaction with synaptic anchoring protein,PSD-95 were compared in synaptosomes from treated mice. NMDAR activationand signaling was initiated by incubation of 6 slices with either 0.3 mMMg²⁺ containing KR (LMKR; basal) or LMKR containing 10 μl NMDA and 1 μMglycine at 37° C. for 30 minutes.

The incubation mixture was aerated with 95% O₂/5% CO₂ every 10 min for 1minute during the stimulation. Ligand stimulation was terminated by theaddition of 1 ml of ice-cold Ca²⁺-free K-R containing mixture of proteinphosphatase inhibitors, 0.5 mM EGTA and 0.1 mM EDTA. After harvesting,tissues were briefly centrifuged and homogenized in 0.25 ml of ice-coldimmunoprecipitation buffer. The homogenates were centrifuged at 1000×gfor 5 minutes (4° C.) and the supernatant (post-mitochondrial fraction)was sonicated for 10 seconds on ice.

The proteins were solubilized in 0.5% digitonin, 0.2% sodium cholate and0.5% NP-40 for 60 minutes at 4° C. with end-over-end rotation. Theresultant lysates were cleared by centrifugation at 50,000×g for 5minutes and diluted with 0.75 ml of immunoprecipitation buffer. Proteinconcentrations were measured by Bradford method (Bio-Rad).

To determine the NMDARs association with PSD-95 as well as NMDARsignaling, the levels of NMDAR subunits, PSD-95 and NMDAR-associatedsignaling molecules were measured in anti-NR1 immunoprecipitates. Inthese studies, brain tissue lysates (100 μg) were immunoprecipitatedovernight (about 18 hours) at 4° C. with 2 μg of immobilized anti-NR1onto covalently conjugated protein A-agarose beads (Pierce-ENDOGEN).Anti-NR1 immunoprecipitates were incubated with 75 μl antigen elutionbuffer (Pierce-ENDOGEN) and 2% SDS for 2 minutes on ice, centrifuged toremove antibody-protein A-agarose complexes and neutralized immediatelywith 10 μl 1.5 M Tris buffer, pH 8.8 followed by addition of 65 μl 2 XPAGE sample buffer and boiled for 5 minutes.

Seventy-five μl of the obtained eluates (50%) were size fractionated on7.5% SDS-PAGE. Proteins were transferred to a nitrocellulose membraneand the levels of various NMDA receptor subunits, PSD-95, signalingproteins were measured using Western blotting with antibodies forPSD-95, nNOS, phospholipase C-71, γPKC, pY⁴⁰²PyK2, pY⁴¹⁶Src orphosphotyrosine. The blots were stripped and re-probed with anti-NR1 toassess the immunoprecipitation efficiency and loading.

IR activation and signaling was initiated by incubation of tissue witheither K-R (basal) or K-R containing 1 nM insulin at 37° C. for 30minutes. The incubation mixture was aerated with 95% O₂/5% CO₂ every 10minutes for 1 minute during the stimulation. Ligand stimulation wasterminated by the addition of 1 ml of ice-cold Ca²⁺-free K-R containingmixture of protein phosphatase inhibitors, 0.5 mM EGTA and 0.1 mM EDTA.

Tissues were briefly centrifuged and homogenized in 0.25 ml of ice-coldimmunoprecipitation buffer. The homogenates were centrifuged at 1000×gfor 5 minutes (4° C.) and the supernatant (post-mitochondrial fraction)was sonicated for 10 seconds on ice. The proteins were solubilized in0.5% digitonin, 0.2% sodium cholate and 0.5% NP-40 for 60 minutes at 4°C. with end-over-end rotation. The resultant lysates were then clearedby centrifugation at 50,000×g for 5 minutes and diluted with 0.75 ml ofimmunoprecipitation buffer. Protein concentrations were measured byBradford method (Bio-Rad).

To determine the IR activation and signaling, the levels ofpY^(1150/1151)IRβ and the level of IR signal transducer, IRS-1 weremeasured in anti-IRβ immunoprecipitates. In these studies, brain tissuelysates (100 μg) were immunoprecipitated overnight (about 18 hours) at4° C. with 2 μg of immobilized anti-IRβ onto covalently conjugatedprotein A-agarose beads (Pierce-ENDOGEN).

Anti-IRβ immunoprecipitates were incubated with 75 ml antigen elutionbuffer (Pierce-ENDOGEN) and 2% SDS for 2 minutes on ice, centrifuged toremove antibody-protein A-agarose complexes and neutralized immediatelywith 10 μl 1.5 M Tris buffer, pH 8.8 followed by addition of 65 μl2×PAGE sample buffer and boiled for 5 minutes.

Seventy-five μl of the obtained eluates (50%) were then sizefractionated on 7.5% SDS-PAGE. Proteins were transferred tonitrocellulose membrane and the levels of pY^(1150/1151)IRβ and IRS-1proteins were measured using Western blotting with antibodies forpY^(1150/1151)IRβ and IRS-1. The blots were stripped and re-probed withanti-IRβ to assess the immunoprecipitation efficiency and loading.

7) Assessment of Cytokine Levels

Parietal cortices (about 10 mg) derived from (1) vehicle-treated sham,(2) compound C0105 treated sham, (3) vehicle-treated ICV Aβ₄₂, and (4)compound C0105 treated ICV Aβ₄₂ mice were first thawed slowly (−80° C.to −20° C. to −4° C.), homogenized in 100 ml of ice-cold homogenizationmedium (25 mM HEPES, pH 7.5; 50 mM NaCl, mixture of protease and proteinphosphatase inhibitors) by sonication and then solubilized with 0.5%polyoxyethylene (40) nonyl phenyl ether (NP-40), 0.2% Na cholate and0.5% digitonin at 4° C. for 1 hour with end-over-end shaking. Followingcentrifugation, the resultant lysate was then dilute with 500 μl (totalvolume 600 μl) and used as the source of cytokines.

To determine the levels of cytokines in these tissues, 0.5 μg/wellbiotinylated mouse monoclonal anti-TNF-α, anti-IL-6 and anti-IL-1β werecoated onto streptavidin-coated plates (Reacti-Bind™ NeutrAvidin™ Highbinding capacity coated 96-well plate; Thermo Scientific Pierce ProteinResearch Products; Rockford, Ill.). Plates were washed 3 times withice-cold 50 mM Tris HCl (pH 7.4) and incubated at 30° C. with 100 μl oflysate derived from the above mentioned tissues for 1 hour.

Plates were washed 3 times with ice-cold 50 mM Tris HCl (pH 7.4) andincubated at 30° C. with 0.5 μg/well un-conjugated rabbit anti-TNF-α,anti-IL-6 and anti-IL-1β for 1 hour. After two washes with 50 mM TrisHCl (pH 7.4), each well was incubated in 0.5 μg/well FITC-conjugatedanti-rabbit IgG (human and mouse absorbed) for 1 hour at 30° C. Plateswere washed twice with 200 μl ice-cold Tris HCl, pH 7.4 and the residualFITC signals were determined by multimode plate reader, DTX880(Beckman). Each lysate was surveyed twice.

8) Immunohistochemical Studies

Quantitative immunohistochemistry on consecutive 5-μm sectionscontaining prefrontal cortex and entorhinal cortex/hippocampus was usedto determine the levels of Aβ₄₂ aggregates/plaques and neurofibrillarypathology (NFT and paired helical filament [PHF] immunoreactivity) usingsingle labeling immunohistochemistry as described previously [[Wang etal., Biol Psychiatry 67:522-530 (2010)]; D'Andrea et azl.,Histopathology 38:120-134 (2001); and Nagele et al., Neuroscience110:199-211 (2002)]. One section was immunostained with anti-NFT or-PHF. The next (consecutive) section (often containing the same neuron)was immunostained with anti-Aβ₄₂ antibodies to measure relative levelsof accumulated Aβ₄₂ peptide in neurons. The relative Aβ₄₂ accumulationextents were compared among different cell types using acomputer-assisted image analysis as described previously [Wang et al., JBiol Chem 275:5626-5632 (2000)].

Brain tissues were fixed at 4° C. in 0.15 M phosphate-buffered 10%formalin, pH 7.4 for 2 weeks, paraffin embedded, serially sectioned at 5μm, and processed for brightfield immunohistochemistry as described. TheAβ₄₂ immunoreactivity was absent when pre-absorbing anti-Aβ₄₂ with Aβ₄₂but not Aβ₄₂₋₁. Specimens were examined using a Nikon FXA microscopewith a Princeton Instruments CCD camera and recorded digitally.

Relative intensities of the NFT/PHF and Aβ₄₂ immunoreactivity weremeasured and compared among similar and different cell types usingImage-Pro® Plus (MediaCybernetics, Inc.; Bethesda, Md.) and Metamorph®software (Molecular Devices, Inc.; Sunnyvale, Calif.) as describedpreviously [D'Andrea et al., Histopathology 38:120-134 (2001)]. Thecorrelations between the amount of NFT/PHF immunoreactivity andAβ₄₂-positive material accumulated within mature neurons were alsodetermined.

Postmortem Tissue

This study protocol conformed to the Declaration of Helsinki: EthicalPrinciples for Biomedical Research Involving Human Beings (the 4^(th)amendment) as reflected in a prior approval by the City College of NewYork and City University of New York Medical School human researchcommittee. The participants had a uniform clinical evaluation thatincluded a medical history, complete neurological examination, cognitivetesting including Mini-Mental state examination and other cognitivetests on episodic memory, semantic memory and language, working memory,perceptual speed, and visuospatial ability as well as psychiatricrating. Based on this information, subjects received AD diagnoses basedon NINCDS-ADRDA criteria [Mckhann et al., Neurology 34, 939-944 (1984)].

Postmortem brain tissues (frontal cortex=FCX) from patients withclinically diagnosed sporadic AD and control tissues from normal,age-matched, neurologically normal individuals were obtained from theHarvard Brain Tissue Resource Center (HBTRC, Belmont, Mass.) and UCLABrain Tissue Resource Center (UBTRC, Los Angeles, Calif.). Both HBTRCand UBTRC are supported in part by Public Health Service grants from theNational Institute of Health. The postmortem time intervals forcollecting these brains were≦13 hours (mean postmortem intervals forcollection of AD and control brain samples were 6.0±0.9 hours and5.8±0.8 hours, respectively).

Diagnostic neuropathological examination was conducted on fixed sectionsstained with hematoxylin and eosin stain and with modified Bielschowskysilver staining [Yamamoto et al., Neuropathol Appl Neurobiol 12, 3-9(1986)] to establish any disease diagnosis according to the criteriadefined by the National Institute on Aging and the Reagan InstituteWorking Group on Diagnostic Criteria for the NeuropathologicalAssessment of AD [Hyman et al., J Neuropathol Exp Neurol 56, 1095-1097(1997)] and brain tissue from age-matched controls was similarlyscreened. The presence of both neuritic (amyloid) plaques andneurofibrillary tangles in all AD brains was confirmed by Nissl andBielschowsky staining as well as characterized immunohisto-chemicallywith anti-Aβ₄₂ and -NFT staining in frontal and entorhinal cortex aswell as hippocampus as described previously ([Wang et al., J Neurochem75, 1155-1161 (2000)].

Control tissues exhibited no gross and minimal, localized microscopicneuropathology of AD (0-3 neuritic plaques/10× field and 0-6 NFTs/10×field in hippocampus). One gram blocks of FCX were dissected using aband saw from fresh frozen coronal brain sections maintained at −80° C.These blocks were derived from Brodmann areas 10 and/or 46. Allpostmortem tissues were identified by an anonymous identificationnumber, and studies were performed as a best matched pair withoutknowledge of clinical information.

The Assessment of Test Compound Effects on Aβ₄₂ Affinity for α7nAChRs

To determine the compound effect on Aβ₄₂ affinity for the α7nAChRs, 200μg of synaptosomes prepared from control subjects were biotinylated. Thebiotinylated synaptosomes were lysed by brief sonication in hypertonicsolutions and used as the tissue source to determine Aβ₄₂ affinity forthe α7nAChRs in the presence and absence of Compound C0105.

In Vitro Treatment of Brain Slices for the Assessment of Test Compoundon α7nAChR-FLNA, TLR4-FLNA and Aβ₄₂-α7nAChR Associations, Ca²⁺ Influx,NMDAR and IR Signaling

Postmortem frontal cortex tissues were gradually thawed (from −80° C. to−20° C.) and were sliced using a chilled Mcllwain tissue chopper (200μm×200 μm×3 mm). Approximately 20 mg of the brain slices were suspendedin 1 ml of ice-cold oxygenated Kreb's-Ringer solution (K-R), containing25 mM HEPES, pH 7.4, 118 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl₂, 1.2 mMKH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, 10 mM glucose, 100 μM ascorbic acid,50 μg/ml leupeptin, 0.2 mM PMSF, 25 μg/ml pepstatin A, and 0.01 U/mlsoybean trypsin inhibitor and centrifuged briefly. Following twoadditional washes with 1 ml of ice-cold K-R, brain slices were suspendedin 1 ml of K-R.

To determine whether exposure to exogenous Aβ₄₂ increases α7nAChR-FLNA,TLR4-FLNA and Aβ₄₂-α7nAChR association and causes Aβ₄₂-induced α7nAChRand N-methyl-D-aspartate receptor (NMDAR) dysfunction, approximately 20mg of frontal cortical slices from control subjects were incubated with0.1 μM of Aβ₄₂ at 37° C. for 1 hour. To test the effects of C0105 onAβ₄₂-incubated control and native AD tissues, Compound C0105 (0.1 and 1nM) was added 10 minutes following 0.1 μM Aβ₄₂. Incubation continued for1 hour in the dark to minimize light destruction of the test agents. Theincubation mixture in a total incubation volume of 0.5 ml was aeratedwith 95% O₂/5% CO₂ every 15 minutes for 1 minute during the incubation.Reaction was terminated by the addition of 1.5 ml of ice-cold Ca²⁺-freeK-R. Tissue slices were harvested by a brief centrifugation and used asthe tissue sources for various assays.

To assess the effects of various α7nAChR agents on α7nAChR-FLNAlinkages, about 20 mg of FCX from control subjects was incubated with 1mM nicotine, PNU282987, α-bungarotoxin, methyllycaconitine, galantamine,memantine, and Aβ₄₀ along with 0.1 μM Aβ₄₂. Incubation continued for 1hour in the dark. The incubation mixture in a total incubation volume of0.5 ml was aerated for 1 minute every 15 minutes with 95% O₂/5% CO₂. Thereaction was terminated by the addition of 1.5 ml of ice-cold Ca²⁺-freeK-R, and slices were collected by a brief centrifugation.

Separately, the compound effect on α7nAChR-FLNA, TLR4-FLNA andAβ₄₂-α7nAChR complex levels were determined after incubation with 0.1and 1 nM compounds in matching Krebs-Ringer and Aβ₄₂-incubatedsynaptosomes from control subjects and Krebs-Ringer incubatedAlzheimer's disease patients. The levels of α7nAChR-FLNA, TLR4-FLNA andAβ₄₂-α7nAChR complexes in the obtained synaptosomes were measured byco-immunoprecipitation method as described below that has been published[Wang et al., J Neurosci 35, 10961-10973 (2009)].

Assessment of α7nAChR-FLNA, TLR4-FLNA and Aβ₄₂-α7nAChR Association byCo-Immunoprecipitation

Two-hundred μg of synaptosomes are pelleted by centrifugation and thensolubilized by brief sonication in 250 μl of immunoprecipitation buffer(25 mM HEPES, pH 7.5; 200 mM NaCl, 1 mM EDTA, 50 μg/ml leupeptin, 10μg/ml aprotinin, 2 μg/ml soybean trypsin inhibitor, 0.04 mM PMSF, 5 mMNaF, 1 mM sodium vanadate, 0.5 mM β-glycerophosphate and 0.1%2-mercaptoethanol containing 0.5% digitonin, 0.2% sodium cholate and0.5% NP-40 and incubated at 4° C. with end-to-end shaking for 1 hour.Following dilution with 750 μl of ice-cold immunoprecipitation bufferand centrifugation (4° C.) to remove insoluble debris, theα7nAChR-/LR4-FLNA and Aβ₄₂-α7nAChR complexes in the lysate are isolatedby immunoprecipitation with 16 hours of incubation at 4° C. withimmobilized rabbit anti-FLNA (1 μg)- and anti-Aβ₄₂ antibodies (1μg)-protein A-conjugated agarose beads, respectively.

The resultant immunocomplexes were pelleted by centrifugation at 4° C.After three washes with 1 ml of ice-cold phosphate-buffered saline (PBS)(pH 7.2) and centrifugation, the isolated α7nAChR-/TLR4-FLNA andAβ₄₂-α7nAChR complexes are solubilized by boiling for 5 minutes in 100μl of SDS-PAGE sample preparation buffer (62.5 mM Tris-HCl, pH 6.8; 10%glycerol, 2% SDS; 5% 2-mercaptoethanol, 0.1% bromophenol blue). Thecontent of α7nAChRs in 50% of the obtained anti-Aβ₄₂ immunoprecipitatewas determined by Western blotting with monoclonal anti-α7nAChRantibodies. In the assay for determining Aβ₄₂-α7nAChR complex level,immobilized rabbit anti-actin (0.5 μg)-protein A-conjugated agarose wereadded together with anti-Aβ₄₂ in the co-immunoprecipitation process.

The content of β-actin in resultant immunoprecipitates is then analyzedby immunoblotting using monoclonal anti-β-actin to illustrate evenimmunoprecipitation efficiency and loading. In the assay for determiningα7nAChR-/TLR4-FLNA complex levels, the blots obtained are stripped andre-probed with monoclonal anti-FLNA for assessing immunoprecipitationefficiency and loading.

Assessment of Ca²⁺ Influx in Synaptosomes as a Functional Measurement ofthe Compounds

NMDAR-, α7nAChR- and voltage-gated calcium channel-mediated [⁴⁵Ca²⁺]influx were studied using synaptosomes prepared from postmortem frontalcortical slices from control and AD subjects. In brief, brainsynaptosomes (100 μg for postmortem study) were incubated at 37° C. for5 minutes in oxygenated 0.3 mM Mg²⁺ K-R (low Mg ²⁺ K-R, LMKR) containing5 μM ⁴⁵Ca²⁺ (10 Ci/mmol) followed by incubation with vehicle, 0.1-10 μMPNU 282987, a specific α7nAChR agonist, or 0.1-10 μM NMDA+1 μM glycinefor 5 minutes or 65 mM K⁺ (made with isomolar replacement of Na⁺) for 1minute in a total incubation volume of 0.5 ml. The reaction wasterminated by addition of 0.5 ml ice-cold Ca²⁺-free K-R containing 0.5mM EGTA and centrifugation at 4° C. After two additional washes, ⁴⁵Ca²⁺contents in synaptosomes were assessed using scintillation spectrometry(Beckman). The background ⁴⁵Ca²⁺ was estimated using hypotonically lysedsynaptosomes. The absolute Ca²⁺ influx was calculated by subtracting thebackground ⁴⁵Ca²⁺ count. The percent increase in Ca²⁺ influx wascalculated as % [(drug-treated-vehicle)/vehicle].

NMDAR Signaling and Association with PSD-95

NMDAR signaling and their interaction with synaptic anchoring protein,PSD-95 were compared in K-R and Compound C0105 (1 nM)-exposed frontalcortical slices from control and AD subjects. NMDAR activation andsignaling were initiated by incubation of approximately 10 mg of invitro treated brain slices with either LMKR (basal) or LMKR containing10 μM NMDA and 1 μM glycine at 37° C. for 30 minutes. The incubationmixture was aerated with 95% O₂/5% CO₂ every 10 minutes for 1 minuteduring the stimulation. Ligand stimulation was terminated by theaddition of 1 ml of ice-cold Ca²⁺-free K-R containing 0.5 mM EGTA and0.1 mM EDTA.

Brain slices were harvested by a brief centrifugation and werehomogenized in 0.25 ml of ice-cold immunoprecipitation buffer. Thehomogenates were centrifuged at 1000×g for 5 minutes (4° C.) and thesupernatant (post-mitochondrial fraction) is sonicated for 10 seconds onice. The proteins are solubilized in 0.5% digitonin, 0.2% sodium cholateand 0.5% NP-40 for 60 minutes at 4° C. with end-over-end rotation. Theresultant lysates are then cleared by centrifugation at 50,000×g for 5minutes and diluted with 0.75 ml of immunoprecipitation buffer. Proteinconcentrations are measured by Bradford method (Bio-Rad).

To determine the NMDAR signaling and the NMDAR complexes associationwith PSD-95 [also known as Disks large homolog 4 (DLH4)], the levels ofNMDAR subunits, PSD-95 and NMDAR-associated signaling molecules weremeasured in anti-NR1 immunoprecipitates. Two NR1 and two NR2 proteinsubunits form the heterotetramer NMDA receptor.

In these studies, brain slice lysates (200 μg) were immunoprecipitatedovernight (about 18 hours) at 4° C. with 2 μg of immobilized anti-NR1onto covalently conjugated protein A-agarose beads (Pierce-ENDOGEN).Anti-NR1 immunoprecipitates were incubated with 75 μl antigen elutionbuffer (Pierce-ENDOGEN) and 2% SDS for 2 minutes on ice, centrifuged toremove antibody-protein A-agarose complexes and neutralized immediatelywith 10 μl 1.5 M Tris buffer, pH 8.8, followed by addition of 65 μl2×PAGE sample buffer and boiled for 5 minutes. Seventy-five μl of theobtained eluates (50%) were then size fractionated on 7.5% SDS-PAGE.Proteins were transferred to nitrocellulose membrane and the levels ofvarious NMDA receptor subunits, PSD-95, signaling proteins were measuredusing Western blotting with antibodies for NR1, PSD-95, nNOS,phospholipase C-γ1, γPKC, pY⁴⁰²PyK2, pY⁴¹⁶Src or phosphotyrosine. Theblots were stripped and re-probed with anti-NR1 or —NR2A/—NR2B to assessthe loading as appropriate.

IR Activation and Signaling

IR signaling was compared in K-R and compound C0105-exposed frontalcortical slices from control and AD subjects. IR activation andsignaling were initiated by incubation of approximately 10 mg of invitro treated brain slices with either KR (basal) or KR containing 1 nMinsulin at 37° C. for 30 minutes. The incubation mixture was aeratedwith 95% O₂/5% CO₂ every 10 minutes for 1 minute during the stimulation.Ligand stimulation was terminated by the addition of 1 ml of ice-coldCa²⁺-free K-R containing 0.5 mM EGTA and 0.1 mM EDTA. Brain slices wereharvested by a brief centrifugation and were homogenized in 0.25 ml ofice-cold immunoprecipitation buffer. The homogenates were centrifuged at1000×g for 5 minutes (4° C.) and the supernatant (post-mitochondrialfraction) is sonicated for 10 seconds on ice. The proteins aresolubilized in 0.5% digitonin, 0.2% sodium cholate and 0.5% NP-40 for 60minutes at 4° C. with end-over-end rotation. The resultant lysates arethen cleared by centrifugation at 50,000×g for 5 minutes and dilutedwith 0.75 ml of immunoprecipitation buffer. Protein concentrations aremeasured by Bradford method (Bio-Rad).

To determine the IR signaling, the levels of pY^(1150/1151)- andpY⁹⁷²-IRs as well as insulin receptor substrate (IRS)-1 recruited to IRwere measured in anti-IRβ immunoprecipitates. In these studies, brainslice lysates (200 μg) were immunoprecipitated overnight (about 18hours) at 4° C. with 2 μg of immobilized anti-IRP onto covalentlyconjugated protein A-agarose beads (Pierce-ENDOGEN). Anti-IRβimmunoprecipitates were incubated with 75 μl antigen elution buffer(Pierce-ENDOGEN) and 2% SDS for 2 minutes on ice, centrifuged to removeantibody-protein A-agarose complexes and neutralized immediately with 10μl 1.5 M Tris buffer, pH 8.8 followed by addition of 65 μl 2 X PAGEsample buffer and boiled for 5 minutes. Seventy-five μl of the obtainedeluates (50%) were then size fractionated on 7.5% SDS-PAGE. Proteinswere transferred to nitrocellulose membrane and the levels of activatedIR (pY^(1150/1151) and pY⁹⁷²) and IRS-1 recruited were measured usingWestern blotting with antibodies for pY^(1150/1151)-IRβ, pY⁹⁷²-IRβ orIRS-1. The blots were stripped and re-probed with anti-IRβ to assess theloading as appropriate.

Western Blot Analysis

Solubilized immunoprecipitates derived from co-immunoprecipitationassays were separated by either 7.5 or 10% SDS-PAGE and thenelectrophoretically transferred to nitrocellulose membranes. Themembranes were washed with PBS and blocked overnight (about 18 hours) at4° C. with 10% milk in PBS with 0.1% Tween®-20 (PBST). Following three5-minute washes with 0.1% PBST, the membranes were incubated at roomtemperature for 2 hours with antibody of choice at 1:500-1:1,000dilutions. After three 2-minute washes in 0.1% PBST, membranes wereincubated for 1 hour with anti-species IgG-HRP (1:5,000 dilution) andwashed with 0.1% PBST three times, 2-minutes each. Immunoreactivity wasvisualized by reacting with chemiluminescent reagent (Pierce-ENDOGEN)for exactly 5 minutes and immediately exposing to X-ray film. Specificbands were quantified by densitometric scanning (GS-800 calibrateddensitometer, Bio-Rad Laboratories).

Assessment of LPS-Induced Tau Phosphorylation Using Postmortem HumanFrontal Cortical Slices

Hippocampi from non-demented control subjects were chopped into coronalslices (100 μm thickness) using a Mcllwain tissue chopper (BrinkmanInstruments) as previously described [Wang et al., J Neurosci,29:10961-10973 (2009)]. The slices are carefully separated in 10 ml ofoxygenated ice-cold dissection medium with two pairs of fine forceps andgentle shaking.

To determine whether Compound C0105 can reduce LPS- and Aβ₄₂-induced tauphosphorylation, approximately 20 mg of human hippocampal slices wereincubated at 37° C. for 1 hour with 1 or 10 μg LPS or 0.1 μM Aβ₄₂ inoxygenated Kreb's-Ringer (25 mM HEPES, pH 7.4; 118 mM NaCl, 4.8 mM KCl,25 mM NaHCO₃, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 10 mM glucose,100 μM ascorbic acid, 50 μg/ml leupeptin, 10 μg/ml aprotinin, 2 μg/mlsoybean trypsin inhibitor, 0.04 mM PMSF and mixture of proteinphosphatase inhibitors) with or without 1 nM Compound C0105 (totalincubation volume is 500 μl). The reaction mixture was aerated with 95%O₂/5% CO₂ for 1 minute every 10 minutes. The reaction was terminated bydiluting with 1.5 ml of ice-cold 1 mM EDTA-containing Ca²⁺-freeKrebs-Ringer and centrifuged for 10 minutes at 15,000×g (4° C.).

After removal of the supernatant, the slices were homogenized to preparepost-mitochondrial (P2) fraction. The obtained P2 fraction wassolubilized in 500 μl of immunoprecipitation buffer (25 mM HEPES, pH7.5;200 mM NaCl, 1 mM EDTA, 0.02% 2-mercaptoethanol, 50 μg/ml leupeptin, 10mg/ml aprotinin, 2 μg/ml soybean trypsin inhibitor, 0.04 mM PMSF andmixture of protein phosphatase inhibitors) containing 0.5% digitonin,0.2% sodium cholate and 0.5% NP-40. Following dilution with 1.5 ml ofice-cold immunoprecipitation buffer and centrifugation (4° C.) to removeinsoluble debris, the total tau proteins in the tissue lysate werepurified by immunoprecipitation with incubating with immobilizedanti-tau antibodies (1 μg) for 16 hours at 4° C. as previously described[Wang et al., J Biol Chem, 278:31547-31553 (2003); Wang et al., BiolPsychiatry, 67:522-530 (2010)].

The anti-tau immunocomplexes were enriched by incubation with 50 μl ofprotein A/G-conjugated agarose beads at 4° C. and centrifugation. Thepurified tau proteins were washed three times with ice-coldphosphate-buffered saline (pH 7.2) and then solubilized by boiling for 5minutes in 150 μl of SDS-PAGE sample preparation buffer (62.5 mMTris-HCl, pH6.8; 10% glycerol, 2% SDS; 5% 2-mercaptoethanol, 0.1%bromophenol blue). The content of phosphorylated serine-202-tau,threonine-231-tau and threonine-181-tau in 50 μl of solubilized anti-tauimmunoprecipitate was separated on 8-16% SDS-PAGE, electrophorecticallytransferred to nitrocellulose membrane and determined by Western blotswith specific antibodies directed against pSerine-202-tau (AT-8),pThreonine-231-tau (AT-180) and pThreonine-181-tau (AT270),respectively.

To ensure equal amounts of tau precipitated by anti-tau antibodies areloaded onto SDS-PAGE, the total tau content in the anti-tauimmunoprecipitate was determined by Western blot using anti-tau antibody(Tau-5) that does not discriminate between phosphorylated andnon-phosphorylated tau.

These results are illustrated in FIGS. 47A, 47B and 47C.

Effects on Release of Pro-Inflammatory Cytokines (IL-1β, IL-6 and TNFα)Induced from Primary Human Astrocytes by Contact with Aβ₄₂ and LPS

Human astrocytes express both the TLR4 and TLR2 cell surface receptors.Aβ₄₂ and LPS each bind to and activate the TLR4 signaling pathwayresulting in the release of pro-inflammatory cytokines such as IL-1β,IL-6 and TNFα, as is shown in previous studies discussed herein, as wellas in the studies shown in FIGS. 48A and 48B.

Experimental Design:

A primary astrocyte culture was prepared according to the provider(Lonza). The adherent astrocytes were trypsinized by 0.25% trypsin-EDTA,then collected and sub-cultured in 12-well plate (1.2 ml/well). When thecells were 80-85% confluent, cells were treated in an incubator under 5%CO₂ with 100 fM, 10 pM or 1 nM Compound C0105 immediately followed bythe addition of Aβ₄₂ (0.1 μM) and LPS (1 μg/ml); i.e., simultaneouslyadding the insulting ligand and Compound C0105 to the cells. Vehiclegroups were treated with 0.1% DMSO only. Incubation continued for 24hours post addition. Culture medium was used as the blank (non-treat)and the levels of cytokines, TNF-α, IL-6 and IL-1β in 200 μl of culturemedium were determined. Each well was sampled once.

To determine the effect of Compound C0105 on cytokine release from humanastrocytes, 0.5 μg/well biotinated mouse monoclonal anti-TNF-α, -IL-6and -IL-1β were coated onto streptavidin-coated plates (Reacti-Bind™NeutrAvidin™ High binding capacity coated 96-well plates). Plates werewashed 3 times with ice-cold 50 mM Tris HCl (pH 7.4) and incubated at30° C. with 200 μl medium derived from the above mentioned conditions.Plates were washed 3 times with ice-cold 50 mM Tris HCl (pH 7.4) andincubated at 30° C. with 0.5 μg/well un-conjugated rabbit anti-TNF-α,-IL-6 and -IL-1β for 1 hour. After three 1 minute washes with 50 mM TrisHCl (pH 7.4), each well was incubated in 0.25 μg/well FITC-conjugatedanti-rabbit IgG (human and mouse absorbed) for 1 hour at 30° C. Plateswere washed twice with 200 μl ice-cold Tris HCl, pH 7.4 and the residualFITC signals were determined by multimode plate reader, DTX880(Beckman).

The results of these studies are shown in FIG. 48 for Aβ₄₂ and LPS. Ascan be seen, Compound C0105 inhibited release of each of the assayedcytokines by about 75 to about 95 percent for each of the threecytokines and each of the four ligands. Statistical analysis by one-wayANOVA: p<0.01; p*<0.01 compared to vehicle treated group for eachinsult.

FLNA Affinity Binding Studies

A series of binding studies using various compounds as ligand and FLNAor the FLNA pentamer of SEQ ID NO:1 as the receptor. These studies werecarried out in a generally similar manner using a competition(displacement) curve for the inhibition of [³H]NLX binding by in thepresence of the ligand, and the results are shown in FIG. 16. Specificsof each study are set out below.

The competition (displacement) curve (FIG. 48A) for the inhibition of[³H]NLX binding by naltrexone to membranes from FLNA-expressing A7(human melanocytic; ATCC CRL-2500) cells that are free of most receptorsand particularly mu shows two affinity states with IC_(50-H) (high) of3.94 picomolar and IC_(50-L) (low) of 834 picomolar. A nonlinearcurve-fit analysis was performed using a competition equation thatassumed two saturable sites for the naltrexone curve comprising of 16concentrations ranging from 0.1 pM to 1 mM. Data are derived from sixstudies each using a different set of A7 cells.

The binding affinity of Compound C0105 for FLNA was similarly determined(FIG. 16B). Briefly, 100 mg of A7 cell membranes were incubated with 0.5nM [³H]NLX in the presence of 0.01 nM-1 mM Compound C0105 at 30° C. for60 minutes in 250 ml of the binding medium (50 mM Tris-HCl, pH 7.5; 100mM NaCl; and protease and protein phosphatase inhibitors). Nonspecificbinding was defined by 1 mM NTX. Reactions were terminated by rapidfiltration through 3% BSA-treated glass microfiber binder free grade B(GF/B) membranes under vacuum. Filters were washed twice with 5 mlice-cold binding medium, and [³H]NLX retained on the filters wasmeasured by liquid scintillation spectrometry. The data obtained wereanalyzed using the GraphPad Software, Inc. (San Diego, Calif.) Prismprogram. Here, an IC_(50-H) of 0.43 picomolar and IC_(50-L) of 226picomolar were determined. N=4.

The binding affinity of Compound C0105 for FLNA was similarly determined(FIG. 16C). Briefly, 200 mg of SK-N-MC (human neuroepithelioma; ATCCHTB-10) cell membranes that contain with both α7nAChR and mu-opioidreceptors were incubated with 0.5 nM [³H]NLX in the presence of 1 mMDAMGO and 0.01 nM-1 mM Compound C0105 at 30° C. for 60 minutes in 250 mlof the binding medium (50 mM Tris-HCl, pH 7.5; 100 mM NaCl; and proteaseand protein phosphatase inhibitors). Nonspecific binding was defined by1 mM NTX. Reactions were terminated by rapid filtration through 3%BSA-treated GF/B membranes under vacuum. Filters were washed twice with5 ml ice-cold binding medium, and [³H]NLX retained on the filters wasmeasured by liquid scintillation spectrometry. The data obtained wereanalyzed using the GraphPad Software, Inc. (San Diego, Calif.) Prismprogram. Here, an IC_(50-H) of 0.201 picomolar and IC_(50-L) of 111picomolar were determined. N=4.

The binding affinity of Compound C0105 for the VAKGL peptide was alsodetermined by a displacement assay (FIG. 16D). Briefly, 10 mg ofN-terminal biotinylated VAKGL (SEQ ID NO:1) peptide (Bn-VAKGL) wasincubated with 0.5 nM [³H]NLX in the presence of 0.01 nM-1 mM CompoundC0105 at 30° C. for 60 minutes in 250 ml of the binding medium (50 mMTris-HCl, pH 7.5; 100 mM NaCl; and protease and protein phosphataseinhibitors). Nonspecific binding was defined by 1 mM NTX. The reactionwas terminated by addition of 1 ml of ice-cold binding medium. The[³H]NLX-bound Bn-VAKGL was trapped by incubation with 20 mlNeutrAvidin®-agarose (Thermo), followed by centrifugation. Following two1.5 ml washes with PBS, the bound [³H]NLX was determined usingscintillation spectrometry. The data obtained were analyzed using theGraphPad Software, Inc. (San Diego, Calif.) Prism program. Here, asingle IC₅₀ value was obtained, as was expected for the 5-mer peptide ofSEQ ID NO:1, and its value was 2.76 picomolar. N=4.

The data obtained in these studies illustrate the similar affinitiesexhibited between naloxone and illustrative Compound C0105 for FLNA.These data also illustrate the similarity in binding activity as areceptor shown between the intact FLNA molecule and the 5-mer FLNApeptide of SEQ ID NO:1, and thereby validate the use of that 5-merpeptide as a surrogate for the complete molecule in the assays carriedout herein.

Each of the patents, patent applications and articles cited herein isincorporated by reference. The use of the article “a” or “an” isintended to include one or more.

The foregoing description and the examples are intended as illustrativeand are not to be taken as limiting. Still other variations within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

1. A method of inhibiting phosphorylation of the tau protein thatcomprises the steps of administering to cells of the central nervoussystem in recognized need an effective amount of a of a compound or apharmaceutically acceptable salt thereof that binds to a pentapeptide offilamin A (FLNA) of SEQ ID NO: 1, inhibits at least about 60 percent ofthe FITC-labeled naloxone binding when present at a 10 μM concentrationand using unlabeled naloxone as the control inhibitor at the sameconcentration, said administration being carried out in the absence of amu opioid receptor- (MOR-) binding effective amount of a separate MORagonist or antagonist.
 2. The method according to claim 1, wherein saidcompound contains at least four of the six pharmacophores of FIGS.35-40.
 3. The method according to claim 1, wherein said compound or apharmaceutically acceptable salt thereof is present dissolved ordispersed in a pharmaceutically acceptable diluent as a pharmaceuticalcomposition when administered.
 4. The method according to claim 1,wherein said compound exhibits less than about 80 percent the MORstimulation provided by DAMGO at the same concentration.
 5. The methodaccording to claim 1, wherein said compound is a compound of Series Athat corresponds in structure to the Formula A below:

wherein R¹ and R² are the same or different and are independentlyselected from the group consisting of H, halogen, C₁-C₁₂ hydrocarbyl,C₁-C₆ acyl, C₁-C₆ hydrocarbyloxy, CF₃ and NR³R⁴, wherein R³ and R⁴ arethe same or different and are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C1-C4hydrocarbylsulfonyl, or R³ and R⁴ together with the depicted nitrogenform a 5-7-membered ring that optionally contains 1 or 2 additionalhetero atoms that independently are nitrogen, oxygen or sulfur; A and Bare the same or different and are CH₂, CDH or CD₂ (where D isdeuterium); X is OH or NR⁵R⁶ wherein R⁵ and R⁶ are the same or differentand are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, orR⁵ and R⁶ together with the depicted nitrogen form a 5-7-membered ringthat optionally contains 1 or 2 additional hetero atoms thatindependently are nitrogen, oxygen or sulfur; R⁷ and R⁸ are the same ordifferent and are H, C₁-C₆ hydrocarbyl, C₁-C₆ acyl, C₁-C₆hydrocarbylsulfonyl, or R⁷ and R⁸ together with the depicted nitrogenform a ring structure W; W contains 5 to 14 atoms in the ring structureincluding the depicted nitrogen, and optionally contains: a) 1 or 2further hetero atoms that are independently oxygen, nitrogen or sulfur,and b) one or more substituent groups bonded to one or more ring atoms,in which the one or more substituents contain a total of up to 8 atomsselected from the group consisting of carbon, nitrogen, oxygen andsulfur, and mixtures thereof; and the dashed line (- - - -) represents1, 2, or 3 optional double bonds.
 6. The method according to claim 5,wherein said compound is a compound of Series A that corresponds instructure to the Formula Ia, below,

wherein R¹ and R² are the same or different and are independently H, orC₁-C₆ hydrocarbyl; A and B are the same or different and are CH₂, CDH orCD₂ (where D is deuterium); W is a ring structure that contains 5 to 14atoms in the ring structure including the depicted nitrogen, thatoptionally contains: a) 1, 2 or 3 further hetero atoms that areindependently oxygen, nitrogen or sulfur, and b) one or more substituentgroups bonded to one or more ring atoms, in which the one or moresubstituents contains a total of up to 14 atoms selected from the groupconsisting of carbon, nitrogen, oxygen and sulfur, and mixtures thereof;and the dashed line (- - - -) represents 1, 2, or 3 optional doublebonds.
 7. The method according to claim 6, wherein said compound is acompound of Series A that corresponds in structure to the Formula II,below,

wherein A, B, W and X and R¹ and R² are as previously defined for acompound of Formula Ia.
 8. The method according to claim 5, wherein saidcompound is a compound of Series A that corresponds in structure to theFormula III, below,

wherein A, B, W and X, R¹ and R² are as previously defined for acompound of Formula I.
 9. The method according to claim 5, wherein saidcompound is a compound of Series A that corresponds in structure to oneor more of the compounds whose formulas are shown below:


10. The method according to claim 1, wherein said compound is a compoundof Series B that corresponds in structure to the Formula I below:

wherein n=0 or 1; m=0 or 1; m+n=0, 1 or 2;

W is an aromatic ring containing 0, 1 or 2 hetero atoms that can benitrogen, oxygen or sulfur, or mixtures thereof in the ring; R¹ isselected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, halogen, cyano, C₁-C₆ hydrocarbyloxyhydrocarboxylene,trifluoromethyl, and hydroxyl; R² is selected from the group consistingof H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆hydrocarbyloxyhydrocarboxylene and halogen; R³ is absent or C₁-C₆hydrocarbyl; R⁴ is C₁-C₆ hydrocarbyl; X⁻=is an anion or is absent whenR³ is absent; the dashed line indicates an optional double bond betweenthe depicted carbon atoms; and the wavy line indicates that the depictedphenyl substituent can be in the Z or E configuration when the optionaldouble bond is present.
 11. The method according to claim 10, whereinsaid compound is a compound of Series B that corresponds in structure tothe Formula II, below,

wherein n=0 or 1; m=0 or 1; m+n=0, 1 or 2;

X⁻=an anion; R¹ is selected from the group consisting of H, C₁-C₆hydrocarbyl, C₁-C₆ hydrocarbyloxy, halogen, cyano, C₁-C₆hydrocarbyloxyhydrocarboxylene, trifluoromethyl, and hydroxyl; R² isselected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, C₁-C₆ hydrocarbyloxyhydrocarboxylene and halogen; thedashed line indicates an optional double bond between the depictedcarbon atoms; and the wavy line indicates that the depicted phenylsubstituent can be in the Z or E configuration when the optional doublebond is present.
 12. The method according to claim 11, wherein saidcompound is a compound of Series B that corresponds in structure to theFormula III, below,

where, n=0 or 1; m=0 or 1; m+n=0, 1 or 2; X⁻=an anion; R¹ is selectedfrom the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy,halogen, cyano, C₁-C₆ hydrocarbyloxyhydrocarboxylene, trifluoromethyl,and hydroxyl; and R² is selected from the group consisting of H, C₁-C₆hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆ hydrocarbyloxyhydrocarboxyleneand halogen.
 13. The method according to claim 12, wherein said anion,X⁻, is selected from the group consisting of phosphate,hydrogenphosphate, dihydrogenphosphate, sulfate, bisulfate, chloride,bromide, iodide, acetate, formate, benzenesulfonate, methanesulfonate,and toluenesulfonate.
 14. The method according to claim 10, wherein saidcompound is a compound of Series B that corresponds in structure to thecompound whose formulas is shown below:


15. The method according to claim 1, wherein said compound is a compoundof Series C-1 that corresponds in structure to the Formula A below:

wherein G and W are selected from the group consisting of NR²⁰, NR⁷,CH₂, S and O, where R⁷ is H, C₁-C₁₂ hydrocarbyl, or C₁-C₁₂ hydrocarboyland R²⁰ is a group X-circle A-R¹ as defined hereinafter; X and Y are thesame or different and are SO₂, C(O), CH₂, CD₂ (where D is deuterium),OC(O), NHC(S), NHC(NH), or NHC(O); Q is CHR⁹ or C(O); Z is CHR¹⁰ orC(O); each of d, e, f and k is either zero or one and the sum of(d+e+f+k)=2; each of m, n and p is zero or one and the sum of m+n+p is 2or 3; circles A and B are the same or different aromatic orheteroaromatic ring systems; R¹ and R² are the same or different andeach can be hydrogen or represent up to three substituents other thanhydrogen that themselves can be the same or different (R^(1a), R^(1b),and R^(1c), and R^(2a), R^(2b), and R^(2c)), each of those six groups,R^(1a-c) and R^(2a-c), is separately selected from the group consistingof H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆hydrocarbyloxycarbonyl, trifluoromethyl, trifluoromethoxy, C₁-C₇hydrocarboyl, hydroxy-, trifluoromethyl- or halogen-substituted C₁-C₇hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl,halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate,carboxamide or sulfonamide, wherein the amido nitrogen in either grouphas the formula NR³R⁴ wherein R³ and R⁴ are the same or different andare H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringedaryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same or different andare H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵and R⁶ together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur; R⁸, R⁹, and R¹⁰ are each H, or two ofR⁸, R⁹, and R¹⁰ are H and one is a C₁-C₈ hydrocarbyl group that isunsubstituted or is substituted with up to three atoms that are the sameor different and are oxygen or nitrogen atoms; R¹¹, R¹², R¹³ and R¹⁴ areall H, or one of the pair R¹¹ and R¹² or the pair R¹³ and R¹⁴ togetherwith the depicted ring form a saturated or unsaturated 6-membered ring,and the other pair are each H, or they are H and D (where D isdeuterium).
 16. The method according to claim 15, wherein said compoundis a compound of Series C-1 that corresponds in structure to the FormulaI below:

wherein X and Y are the same or different and are SO₂, C(O), CH₂, CD₂(where D is deuterium), NHC(NH), OC(O), NHC(S) or NHC(O); W is NR⁷, CH₂,S or O, where R⁷ is H, C₁-C₁₂ hydrocarbyl, or C₁-C₁₂ hydrocarboyl(acyl); Q is CHR⁹ or C(O); Z is CHR¹⁰ or C(O); J and F are the same ordifferent and are CH or CD (where D is deuterium); each of m, n and p iszero or one and the sum of m+n+p is 2 or 3; and circles A and B are thesame or different aromatic or heteroaromatic ring systems that containone ring or two fused rings; R¹ and R² are the same or different andeach can be hydrogen or represent up to three substituents other thanhydrogen that themselves can be the same or different (R^(1a), R^(1b),and R^(1c), and R^(2a), R^(2b), and R^(2c)) each of those six groups,R^(1a-c) and R^(2a-c), is separately selected from the group consistingof H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, trifluoromethyl,trifluoromethoxy, C₁-C₇ hydrocarboyl, hydroxy-, trifluoromethyl- orhalogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl,halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate,carboxamide or sulfonamide wherein the amido nitrogen of either grouphas the formula NR³R⁴ wherein R³ and R⁴ are the same or different andare H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is where M is —CH₂—, —O— or —N═N— and Ar is asingle-ringed aryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same ordifferent and are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄hydrocarbylsulfonyl, or R⁵ and R⁶ together with the depicted nitrogenform a 5-7-membered ring that optionally contains 1 or 2 additionalhetero atoms that independently are nitrogen, oxygen or sulfur; R⁸, R⁹,and R¹⁰ are each H, or two of R⁸, R⁹, and R¹⁰ are H and one is a C₁-C₈hydrocarbyl group that is unsubstituted or is substituted with up tothree atoms that are the same or different and are oxygen or nitrogenatoms; R¹¹, R¹², R¹³ and R¹⁴ are all H, or R¹¹ and R¹³ are H and R¹² andR¹⁴ are H or D (where D is deuterium), or one of the pair R¹¹ and R¹² orthe pair R¹³ and R¹⁴ together with the depicted ring form a saturated orunsaturated 6-membered ring, and the other pair are each H or they are Hand D.
 17. The method according to claim 15, wherein said compound is acompound of Series C-1 that corresponds in structure to the Formula IIbelow:

wherein Q is CHR⁹ or C(O); Z is CHR¹⁰ or C(O); each of m, n and p iszero or one and the sum of m+n+p is 2 or 3; J and F are the same ordifferent and are CH₂, CHD or CD₂ (where D is deuterium); circles A andB are the same or different aromatic or heteroaromatic ring systems; R¹and R² are the same or different and each can be hydrogen or representup to three substituents other than hydrogen that themselves can be thesame or different (R^(1a), R^(1b), and R^(1c), and R^(2a), R^(2b), andR^(2c)), each of those six groups, R^(1a-c) and R^(2a-c), is separatelyselected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, C₁-C₆ hydrocarbyloxycarbonyl, trifluoromethyl,trifluoromethoxy, C₁-C₇ hydrocarboyl, hydroxy-, trifluoromethyl- orhalogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₅hydrocarbyloxysulfonyl, halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇hydrocarbyl carboxylate, carboxamide or sulfonamide, wherein the amidonitrogen in either group has the formula NR³R⁴ wherein R³ and R⁴ are thesame or different and are H, C₁-C₄ hydrocarbyl, or R³ and R⁴ togetherwith the depicted nitrogen form a 5-7-membered ring that optionallycontains 1 or 2 additional hetero atoms that independently are nitrogen,oxygen or sulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is asingle-ringed aryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same ordifferent and are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄hydrocarbylsulfonyl, or R⁵ and R⁶ together with the depicted nitrogenform a 5-7-membered ring that optionally contains 1 or 2 additionalhetero atoms that independently are nitrogen, oxygen or sulfur.
 18. Themethod according to claim 15, wherein said compound is a compound ofSeries C-1 that corresponds in structure to the Formula III below:

wherein Q is CHR⁹ or C(O); Z is CHR¹⁰ or C(O); each of m, n and p iszero or one and the sum of m+n+p is 2 or 3; J and F are the same ordifferent and are CH₂, CHD or CD2 (where D is deuterium); X and Y areboth CO, or X and Y are different and are SO₂, C(O), CH₂, CD₂, NHC(NH),NHC(S) or NHC(O); circles A and B are the same or different aromatic orheteroaromatic ring systems; R¹ and R² are the same or different andeach can be hydrogen or represent up to three substituents other thanhydrogen that themselves can be the same or different (R^(1a), R^(1b),and R^(1c), and R^(2a), R^(2b), and R^(2c)), each of those six groups,R^(1a-c) and R^(2a-c), is separately selected from the group consistingof H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆hydrocarbyloxycarbonyl, trifluoromethyl, trifluoromethoxy, C₁-C₇hydrocarboyl, hydroxy-, trifluoromethyl- or halogen-substituted C₁-C₇hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl,halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate,carboxamide or sulfonamide, wherein the amido nitrogen in either grouphas the formula NR³R⁴ wherein R³ and R⁴ are the same or different andare H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringedaryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same or different andare H, O₁—C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵and R⁶ together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur.
 19. The method according to claim 15,wherein said compound is a compound of Series C-1 that corresponds instructure to the Formula IV below:

wherein Q is CHR⁹ or C(O); Z is CHR¹⁰ or C(O); each of m, n and p iszero or one and the sum of m+n+p is 2 or 3; J and F are the same ordifferent and are CH₂, CHD or CD₂ (where D is deuterium); X and Y arethe same or different and are SO₂, C(O), CH₂, CD₂, OC(O), NHC(NH),NHC(S) or NHC(O); circles A and B are the same or different aromatic orheteroaromatic ring systems; R¹ and R² are the same or different andeach can be hydrogen or represent up to three substituents other thanhydrogen that themselves can be the same or different (R^(1a), R^(1b),and R^(1c), and R^(2a), R^(2b), and R^(2c)), each of those six groups,R^(1a-c) and R^(2a-c), is separately selected from the group consistingof H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆hydrocarbyloxycarbonyl, trifluoromethyl, trifluoromethoxy, C₁-C₇hydrocarboyl, hydroxy-, trifluoromethyl- or halogen-substituted C₁-C₇hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl,halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate,carboxamide or sulfonamide, wherein the amido nitrogen in either grouphas the formula NR³R⁴ wherein R³ and R⁴ are the same or different andare H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringedaryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same or different andare H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵and R⁶ together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur.
 20. The method according to claim 15,wherein said compound is a compound of Series C-1 that corresponds instructure to the compound whose formulas is shown below:


21. The method according to claim 1, wherein said compound is a compoundof Series C-1 that corresponds in structure to the Formula A below:

wherein Q is CHR⁹ or C(O), Z is CHR¹⁰ or C(O), and only one of Q and Zis C(O); each of m, n and p is zero or one and the sum of m+n+p is 2 or3; each of G, P and W is selected from the group consisting of NR²⁰,NR², NR⁷, S and O, where R⁷ and R² are the same or different and are H,C(H)_(v)(D)_(h) where each of v and h is 0, 1, 2 or 3 and v+h=3,C(H)_(q)(D)_(r)-aliphatic C₁-C₁₁ hydrocarbyl where each of q and r is 0,1, or 2 and q+r=0, 1 or 2, aliphatic C₁-C₁₂ hydrocarbyl sulfonyl oraliphatic C₁-C₁₂ hydrocarboyl, and R²⁰ is X-circle A-R¹ as definedhereinafter; each of d, e, f and k is either zero or one and the sum of(d+e+f+k)=2; J and F are the same or different and are CH or CD (where Dis deuterium); E and K are the same or different and are CH₂, CHD or CD₂(where D is deuterium); X is SO₂, C(O), CH₂, CD₂, OC(O), NHC(NH), NHC(S)or NHC(O); circle A is an aromatic or heteroaromatic ring system thatcontains a single ring or two fused rings; R¹ is H or represents up tothree substituents, R^(1a), R^(1b), and R^(1c), that themselves can bethe same or different, wherein each of those three groups, R^(1a-c), isseparately selected from the group consisting of H, C₁-C₆ hydrocarbyl,C₁-C₆ hydrocarbyloxy, C₁-C₆ hydrocarbyloxycarbonyl, trifluoromethyl,trifluoromethoxy, C₁-C₇ hydrocarboyl, hydroxy-, trifluoromethyl- orhalogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₆hydrocarbyloxysulfonyl, halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇hydrocarbyl carboxylate, carboxamide or sulfonamide, wherein the amidonitrogen in either amide group has the formula NR³R⁴ in which R³ and R⁴are the same or different and are H, C₁-C₄ hydrocarbyl, or R³ and R⁴together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur, MAr, where M is —CH₂—, —O— or —N═N— andAr is a single-ringed aryl or heteroaryl group and NR⁵R⁶ wherein R⁵ andR⁶ are the same or different and are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl,C₁-C₄ hydrocarbylsulfonyl, or R⁵ and R⁶ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur; R⁸, R⁹, and R¹⁰ are each H, or two of R⁸, R⁹, and R¹⁰ are H andone is a C₁-C₈ hydrocarbyl group that is unsubstituted or is substitutedwith up to three atoms that are the same or different and are oxygen ornitrogen atoms; R¹¹, R¹², R¹³ and R¹⁴ are all H, or R¹¹ and R¹³ are Hand R¹² and R¹⁴ are H or D (where D is deuterium), or one of the pairR¹¹ and R¹² or the pair R¹³ and R¹⁴ together with the depicted ring forma saturated or unsaturated 6-membered ring, and the other pair are eachH or they are H and D.
 22. The method according to claim 21, whereinsaid compound is a compound of Series C-2 that corresponds in structureto the Formula I below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium).
 23. The method according to claim 21, wherein saidcompound is a compound of Series C-2 that corresponds in structure tothe Formula II below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium).
 24. The method according to claim 21, wherein saidcompound is a compound of Series C-2 that corresponds in structure tothe Formula III below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium); and each of m and n is one.
 25. The method according toclaim 21, wherein said compound is a compound of Series C-2 thatcorresponds in structure to the Formula IV below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium).
 26. The method according to claim 21, wherein saidcompound is a compound of Series C-2 that corresponds in structure tothe Formula V below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium).
 27. The method according to claim 21, wherein saidcompound is a compound of Series C-2 that corresponds in structure tothe Formula VI below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium); and each of m and n is one.
 28. The method according toclaim 21, wherein said compound is a compound of Series C-2 thatcorresponds in structure to the compound whose formulas is shown below:


29. The method according to claim 1, wherein said administration iscarried out a plurality of times.
 30. The method according to claim 29,wherein said administration is carried out daily.
 31. The methodaccording to claim 29, wherein said administration is carried outmultiple times daily.
 32. The method according to claim 1, wherein saidcompound or the pharmaceutically acceptable salt of said compound isadministered in a pharmaceutical composition.
 33. The method accordingto claim 1, wherein said pharmaceutical composition is in liquid form.34. The method according to claim 33, wherein said pharmaceuticalcomposition is in solid form.
 35. A method of inhibiting a TLR4-mediatedimmune response that comprises administering to TLR4-containing cells inrecognized need thereof an effective amount of a compound or apharmaceutically acceptable salt thereof that binds to a pentapeptide offilamin A (FLNA) of SEQ ID NO: 1, inhibits at least about 60 percent andmore preferably about 70 percent of the FITC-labeled naloxone bindingwhen present at a 10 μM concentration and using unlabeled naloxone asthe control inhibitor at the same concentration, and contains at leastfour of the six pharmacophores of FIGS. 35-40, said administration beingcarried out in the absence of a mu opioid receptor- (MOR-) bindingeffective amount of a separate MOR agonist or antagonist.
 36. The methodaccording to claim 35, wherein said compound contains at least five ofthe six pharmacophores of FIGS. 35-40.
 37. The method according to claim35, wherein said compound or a pharmaceutically acceptable salt thereofis present dissolved or dispersed in a pharmaceutically acceptablediluent as a pharmaceutical composition when administered.
 38. Themethod according to claim 35, wherein said compound exhibits less thanabout 80 percent the MOR stimulation provided by DAMGO at the sameconcentration.
 39. The method according to claim 35, wherein saidcompound is a compound of Series A that corresponds in structure to theFormula A below:

wherein R¹ and R² are the same or different and are independentlyselected from the group consisting of H, halogen, C₁-C₁₂ hydrocarbyl,C₁-C₆ acyl, C₁-C₆ hydrocarbyloxy, CF₃ and NR³R⁴, wherein R³ and R⁴ arethe same or different and are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄hydrocarbylsulfonyl, or R³ and R⁴ together with the depicted nitrogenform a 5-7-membered ring that optionally contains 1 or 2 additionalhetero atoms that independently are nitrogen, oxygen or sulfur; A and Bare the same or different and are CH₂, CDH or CD₂; X is OH or NR⁵R⁶wherein R⁵ and R⁶ are the same or different and are H, C₁-C₄hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵ and R⁶together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur; R⁷ and R⁸ are the same or different andare H, C₁-C₆ hydrocarbyl, C₁-C₆ acyl, C₁-C₆ hydrocarbylsulfonyl, or R⁷and R⁸ together with the depicted nitrogen form a ring structure W; Wcontains 5 to 14 atoms in the ring structure including the depictednitrogen, and optionally contains: a) 1 or 2 further hetero atoms thatare independently oxygen, nitrogen or sulfur, and b) one or moresubstituent groups bonded to one or more ring atoms, in which the one ormore substituents contain a total of up to 8 atoms selected from thegroup consisting of carbon, nitrogen, oxygen and sulfur, and mixturesthereof; and the dashed line (- - - -) represents 1, 2, or 3 optionaldouble bonds.
 40. The method according to claim 39, wherein saidcompound is a compound of Series A that corresponds in structure to theFormula Ia, below,

wherein R¹ and R² are the same or different and are independently H, orC₁-C₆ hydrocarbyl; A and B are the same or different and are CH₂, CDH orCD₂; W is a ring structure that contains 5 to 14 atoms in the ringstructure including the depicted nitrogen, that optionally contains: a)1, 2 or 3 further hetero atoms that are independently oxygen, nitrogenor sulfur, and b) one or more substituent groups bonded to one or morering atoms, in which the one or more substituents contains a total of upto 14 atoms selected from the group consisting of carbon, nitrogen,oxygen and sulfur, and mixtures thereof; and the dashed line (- - - -)represents 1, 2, or 3 optional double bonds.
 41. The method according toclaim 40, wherein said compound is a compound of Series A thatcorresponds in structure to the Formula II, below,

wherein A, B, W and X and R¹ and R² are as previously defined for acompound of Formula Ia.
 42. The method according to claim 39, whereinsaid compound is a compound of Series A that corresponds in structure tothe Formula III, below,

wherein A, B, W and X, R¹ and R² are as previously defined for acompound of Formula I.
 43. The method according to claim 39, whereinsaid compound is a compound of Series A that corresponds in structure toone or more of the compounds whose formulas are shown below:


44. The method according to claim 35, wherein said compound is acompound of Series B that corresponds in structure to the Formula Ibelow:

wherein n=0 or 1; m=0 or 1; m+n=0, 1 or 2;

W is an aromatic ring containing 0, 1 or 2 hetero atoms that can benitrogen, oxygen or sulfur, or mixtures thereof in the ring; R¹ isselected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, halogen, cyano, C₁-C₆ hydrocarbyloxyhydrocarboxylene,trifluoromethyl, and hydroxyl; R² is selected from the group consistingof H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆hydrocarbyloxyhydrocarboxylene and halogen; R³ is absent or C₁-C₆hydrocarbyl; R⁴ is C₁-C₆ hydrocarbyl; X⁻=is an anion or is absent whenR³ is absent; the dashed line indicates an optional double bond betweenthe depicted carbon atoms; and the wavy line indicates that the depictedphenyl substituent can be in the Z or E configuration when the optionaldouble bond is present.
 45. The method according to claim 44, whereinsaid compound is a compound of Series B that corresponds in structure tothe Formula II, below,

wherein n=0 or 1; m=0 or 1; m+n=0, 1 or 2;

X⁻=an anion; R¹ is selected from the group consisting of H, C₁-C₆hydrocarbyl, C₁-C₆ hydrocarbyloxy, halogen, cyano, C₁-C₆hydrocarbyloxyhydrocarboxylene, trifluoromethyl, and hydroxyl; R² isselected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, C₁-C₆ hydrocarbyloxyhydrocarboxylene and halogen; thedashed line indicates an optional double bond between the depictedcarbon atoms; and the wavy line indicates that the depicted phenylsubstituent can be in the Z or E configuration when the optional doublebond is present.
 46. The method according to claim 45, wherein saidcompound is a compound of Series B that corresponds in structure to theFormula III, below,

where, n=0 or 1; m=0 or 1; m+n=0, 1 or 2; X⁻=an anion; R¹ is selectedfrom the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy,halogen, cyano, C₁-C₆ hydrocarbyloxyhydrocarboxylene, trifluoromethyl,and hydroxyl; and R² is selected from the group consisting of H, C₁-C₆hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆ hydrocarbyloxyhydrocarboxyleneand halogen.
 47. The method according to claim 46, wherein said anion,X⁻, is selected from the group consisting of phosphate,hydrogenphosphate, dihydrogenphosphate, sulfate, bisulfate, chloride,bromide, iodide, acetate, formate, benzenesulfonate, methanesulfonate,and toluenesulfonate.
 48. The method according to claim 46, wherein saidcompound is a compound of Series B that corresponds in structure to thecompound whose formulas is shown below:


49. The method according to claim 35, wherein said compound is acompound of Series C-1 that corresponds in structure to the Formula Abelow:

wherein G and W are selected from the group consisting of NR²⁰, NR⁷,CH₂, S and O, where R⁷ is H, C₁-C₁₂ hydrocarbyl, or C₁-C₁₂ hydrocarboyland R²⁰ is a group X-circle A-R¹ as defined hereinafter; X and Y are thesame or different and are SO₂, C(O), CH₂, CD₂ (where D is deuterium),OC(O), NHC(S), NHC(NH), or NHC(O); Q is CHR⁹ or C(O); Z is CHR¹⁰ orC(O); each of d, e, f and k is either zero or one and the sum of(d+e+f+k)=2; each of m, n and p is zero or one and the sum of m+n+p is 2or 3; circles A and B are the same or different aromatic orheteroaromatic ring systems; R¹ and R² are the same or different andeach can be hydrogen or represent up to three substituents other thanhydrogen that themselves can be the same or different (R^(1a), R^(1b),and R^(1c), and R^(2a), R^(2b), and R^(2c)), each of those six groups,R^(1a-c) and R^(2a-c), is separately selected from the group consistingof H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆hydrocarbyloxycarbonyl, trifluoromethyl, trifluoromethoxy, C₁-C₇hydrocarboyl, hydroxy-, trifluoromethyl- or halogen-substituted C₁-C₇hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl,halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate,carboxamide or sulfonamide, wherein the amido nitrogen in either grouphas the formula NR³R⁴ wherein R³ and R⁴ are the same or different andare H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringedaryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same or different andare H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵and R⁶ together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur; R⁸, R⁹, and R¹⁰ are each H, or two ofR⁸, R⁹, and R¹⁰ are H and one is a C₁-C₈ hydrocarbyl group that isunsubstituted or is substituted with up to three atoms that are the sameor different and are oxygen or nitrogen atoms; R¹¹, R¹², R¹³ and R¹⁴ areall H, or one of the pair R¹¹ and R¹² or the pair R¹³ and R¹⁴ togetherwith the depicted ring form a saturated or unsaturated 6-membered ring,and the other pair are each H, or they are H and D (where D isdeuterium).
 50. The method according to claim 49, wherein said compoundis a compound of Series C-1 that corresponds in structure to the FormulaI below:

wherein X and Y are the same or different and are SO₂, C(O), CH₂, CD₂(where D is deuterium), NHC(NH), OC(O), NHC(S) or NHC(O); W is NR⁷, CH₂,S or O, where R⁷ is H, C₁-C₁₂ hydrocarbyl, or C₁-C₁₂ hydrocarboyl(acyl); Q is CHR⁹ or C(O); Z is CHR¹⁰ or C(O); J and F are the same ordifferent and are CH or CD (where D is deuterium); each of m, n and p iszero or one and the sum of m+n+p is 2 or 3; and circles A and B are thesame or different aromatic or heteroaromatic ring systems that containone ring or two fused rings; R¹ and R² are the same or different andeach can be hydrogen or represent up to three substituents other thanhydrogen that themselves can be the same or different (R^(1a), R^(1b),and R^(1c), and R^(2a), R^(2b), and R^(2c)) each of those six groups,R^(1a-c) and R^(2a-c), is separately selected from the group consistingof H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, trifluoromethyl,trifluoromethoxy, C₁-C₇ hydrocarboyl, hydroxy-, trifluoromethyl- orhalogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl,halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate,carboxamide or sulfonamide wherein the amido nitrogen of either grouphas the formula NR³R⁴ wherein R³ and R⁴ are the same or different andare H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is where M is —CH₂—, —O— or —N═N— and Ar is asingle-ringed aryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same ordifferent and are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄hydrocarbylsulfonyl, or R⁵ and R⁶ together with the depicted nitrogenform a 5-7-membered ring that optionally contains 1 or 2 additionalhetero atoms that independently are nitrogen, oxygen or sulfur; R⁸, R⁹,and R¹⁰ are each H, or two of R⁸, R⁹, and R¹⁰ are H and one is a C₁-C₈hydrocarbyl group that is unsubstituted or is substituted with up tothree atoms that are the same or different and are oxygen or nitrogenatoms; R¹¹, R¹², R¹³ and R¹⁴ are all H, or R¹¹ and R¹³ are H and R¹² andR¹⁴ are H or D (where D is deuterium), or one of the pair R¹¹ and R¹² orthe pair R¹³ and R¹⁴ together with the depicted ring form a saturated orunsaturated 6-membered ring, and the other pair are each H or they are Hand D.
 51. The method according to claim 49, wherein said compound is acompound of Series C-1 that corresponds in structure to the Formula IIbelow:

wherein Q is CHR⁹ or C(O); Z is CHR¹⁰ or C(O); each of m, n and p iszero or one and the sum of m+n+p is 2 or 3; J and F are the same ordifferent and are CH₂, CHD or CD₂ (where D is deuterium); circles A andB are the same or different aromatic or heteroaromatic ring systems; R¹and R² are the same or different and each can be hydrogen or representup to three substituents other than hydrogen that themselves can be thesame or different (R^(1a), R^(1b), and R^(1c), and R^(2a), R^(2b), andR^(2c)), each of those six groups, R^(1a-c) and R^(2a-c), is separatelyselected from the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆hydrocarbyloxy, C₁-C₆ hydrocarbyloxycarbonyl, trifluoromethyl,trifluoromethoxy, C₁-C₇ hydrocarboyl, hydroxy-, trifluoromethyl- orhalogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₆hydrocarbyloxysulfonyl, halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇hydrocarbyl carboxylate, carboxamide or sulfonamide, wherein the amidonitrogen in either group has the formula NR³R⁴ wherein R³ and R⁴ are thesame or different and are H, C₁-C₄ hydrocarbyl, or R³ and R⁴ togetherwith the depicted nitrogen form a 5-7-membered ring that optionallycontains 1 or 2 additional hetero atoms that independently are nitrogen,oxygen or sulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is asingle-ringed aryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same ordifferent and are H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄hydrocarbylsulfonyl, or R⁵ and R⁶ together with the depicted nitrogenform a 5-7-membered ring that optionally contains 1 or 2 additionalhetero atoms that independently are nitrogen, oxygen or sulfur.
 52. Themethod according to claim 49, wherein said compound is a compound ofSeries C-1 that corresponds in structure to the Formula III below:

wherein Q is CHR⁹ or C(O); Z is CHR¹⁰ or C(O); each of m, n and p iszero or one and the sum of m+n+p is 2 or 3; J and F are the same ordifferent and are CH₂, CHD or CD₂ (where D is deuterium); X and Y areboth CO, or X and Y are different and are SO₂, C(O), CH₂, CD₂ (where Dis deuterium), NHC(NH), NHC(S) or NHC(O); circles A and B are the sameor different aromatic or heteroaromatic ring systems; R¹ and R² are thesame or different and each can be hydrogen or represent up to threesubstituents other than hydrogen that themselves can be the same ordifferent (R^(1a), R^(1b), and R^(1c), and R^(2a), R^(2b), and R^(2c)),each of those six groups, R^(1a-c) and R^(2a-c), is separately selectedfrom the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy,C₁-C₆ hydrocarbyloxycarbonyl, trifluoromethyl, trifluoromethoxy, C₁-C₇hydrocarboyl, hydroxy-, trifluoromethyl- or halogen-substituted C₁-C₇hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl,halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate,carboxamide or sulfonamide, wherein the amido nitrogen in either grouphas the formula NR³R⁴ wherein R³ and R⁴ are the same or different andare H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringedaryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same or different andare H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵and R⁶ together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur.
 53. The method according to claim 49,wherein said compound is a compound of Series C-1 that corresponds instructure to the Formula IV below:

wherein Q is CHR⁹ or C(O); Z is CHR¹⁰ or C(O); each of m, n and p iszero or one and the sum of m+n+p is 2 or 3; J and F are the same ordifferent and are CH₂, CHD or CD2 (where D is deuterium); X and Y arethe same or different and are SO₂, C(O), CH₂, CD₂ (where D isdeuterium), OC(O), NHC(NH), NHC(S) or NHC(O); circles A and B are thesame or different aromatic or heteroaromatic ring systems; R¹ and R² arethe same or different and each can be hydrogen or represent up to threesubstituents other than hydrogen that themselves can be the same ordifferent (R^(1a), R^(1b), and R^(1c), and R^(2a), R^(2b), and R^(2c)),each of those six groups, R^(1a-c) and R^(2a-c), is separately selectedfrom the group consisting of H, C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy,C₁-C₆ hydrocarbyloxycarbonyl, trifluoromethyl, trifluoromethoxy, C₁-C₇hydrocarboyl, hydroxy-, trifluoromethyl- or halogen-substituted C₁-C₇hydrocarboyl, C₁-C₆ hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl,halogen, nitro, phenyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate,carboxamide or sulfonamide, wherein the amido nitrogen in either grouphas the formula NR³R⁴ wherein R³ and R⁴ are the same or different andare H, C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depictednitrogen form a 5-7-membered ring that optionally contains 1 or 2additional hetero atoms that independently are nitrogen, oxygen orsulfur, MAr, where M is —CH₂—, —O— or —N═N— and Ar is a single-ringedaryl group, and NR⁵R⁶ wherein R⁵ and R⁶ are the same or different andare H, C₁-C₄ hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵and R⁶ together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur.
 54. The method according to claim 49,wherein said compound is a compound of Series C-1 that corresponds instructure to the compound whose formulas is shown below:


55. The method according to claim 35, wherein said compound is acompound of Series C-1 that corresponds in structure to the Formula Abelow:

wherein Q is CHR⁹ or C(O), Z is CHR¹⁰ or C(O), and only one of Q and Zis C(O); each of m, n and p is zero or one and the sum of m+n+p is 2 or3; each of G, P and W is selected from the group consisting of NR²⁰,NR², NR⁷, S and O, where R⁷ and R² are the same or different and are H,C(H)_(v)(D)_(h) (where D is deuterium) where each of v and h is 0, 1, 2or 3 and v+h=3, C(H)_(g)(D)_(r)-aliphatic C₁-C₁₁ hydrocarbyl where eachof q and r is 0, 1, or 2 and q+r=0, 1 or 2, aliphatic C₁-C₁₂ hydrocarbylsulfonyl or aliphatic C₁-C₁₂ hydrocarboyl, and R²⁰ is X-circle A-R¹ asdefined hereinafter; each of d, e, f and k is either zero or one and thesum of (d+e+f+k)=2; J and F are the same or different and are CH or CD(where D is deuterium); E and K are the same or different and are CH₂,CHD or CD₂ (where D is deuterium); X is SO₂, C(O), CH₂, CD₂, OC(O),NHC(NH), NHC(S) or NHC(O); circle A is an aromatic or heteroaromaticring system that contains a single ring or two fused rings; R¹ is H orrepresents up to three substituents, R^(1a), R^(1b), and R^(1c), thatthemselves can be the same or different, wherein each of those threegroups, R^(1a-c), is separately selected from the group consisting of H,C₁-C₆ hydrocarbyl, C₁-C₆ hydrocarbyloxy, C₁-C₆ hydrocarbyloxycarbonyl,trifluoromethyl, trifluoromethoxy, C₁-C₇ hydrocarboyl, hydroxy-,trifluoromethyl- or halogen-substituted C₁-C₇ hydrocarboyl, C₁-C₆hydrocarbylsulfonyl, C₁-C₆ hydrocarbyloxysulfonyl, halogen, nitro,phenyl, cyano, carboxyl, C₁-C₇ hydrocarbyl carboxylate, carboxamide orsulfonamide, wherein the amido nitrogen in either amide group has theformula NR³R⁴ in which R³ and R⁴ are the same or different and are H,C₁-C₄ hydrocarbyl, or R³ and R⁴ together with the depicted nitrogen forma 5-7-membered ring that optionally contains 1 or 2 additional heteroatoms that independently are nitrogen, oxygen or sulfur, MAr, where M is—CH₂—, —O— or —N═N— and Ar is a single-ringed aryl or heteroaryl groupand NR⁵R⁶ wherein R⁵ and R⁶ are the same or different and are H, C₁-C₄hydrocarbyl, C₁-C₄ acyl, C₁-C₄ hydrocarbylsulfonyl, or R⁵ and R⁶together with the depicted nitrogen form a 5-7-membered ring thatoptionally contains 1 or 2 additional hetero atoms that independentlyare nitrogen, oxygen or sulfur; R⁸, R⁹, and R¹⁰ are each H, or two ofR⁸, R⁹, and R¹⁰ are Hand one is a C₁-C₈ hydrocarbyl group that isunsubstituted or is substituted with up to three atoms that are the sameor different and are oxygen or nitrogen atoms; R¹¹, R¹², R¹³ and R¹⁴ areall H, or R¹¹ and R¹³ are H and R¹² and R¹⁴ are H or D (where D isdeuterium), or one of the pair R¹¹ and R¹² or the pair R¹³ and R¹⁴together with the depicted ring form a saturated or unsaturated6-membered ring, and the other pair are each H or they are H and D. 56.The method according to claim 55, wherein said compound is a compound ofSeries C-2 that corresponds in structure to the Formula I below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium).
 57. The method according to claim 55, wherein saidcompound is a compound of Series C-2 that corresponds in structure tothe Formula II below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium).
 58. The method according to claim 55, wherein saidcompound is a compound of Series C-2 that corresponds in structure tothe Formula III below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium); and each of m and n is one.
 59. The method according toclaim 55, wherein said compound is a compound of Series C-2 thatcorresponds in structure to the Formula IV below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium).
 60. The method according to claim 55, wherein saidcompound is a compound of Series C-2 that corresponds in structure tothe Formula V below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium).
 61. The method according to claim 55, wherein saidcompound is a compound of Series C-2 that corresponds in structure tothe Formula VI below:

wherein J and F are the same or different and are CH₂, CHD or CD₂ (whereD is deuterium); and each of m and n is one.
 62. The method according toclaim 55, wherein said compound is a compound of Series C-2 thatcorresponds in structure to the compound whose formulas is shown below:


63. The method according to claim 35, wherein said administration iscarried out a plurality of times.
 64. The method according to claim 63,wherein said administration is carried out daily.
 65. The methodaccording to claim 63, wherein said administration is carried outmultiple times daily.
 66. The method according to claim 35, wherein saidcompound or the pharmaceutically acceptable salt of said compound isadministered in a pharmaceutical composition.
 67. The method accordingto claim 66, wherein said pharmaceutical composition is in liquid form.68. The method according to claim 66, wherein said pharmaceuticalcomposition is in solid form.